Phenylpiperazine proprotein convertase subtilisin/kexin type 9 (pcsk9) modulators and their use

ABSTRACT

This invention is related to the field of PCSK9 biology and the composition and methods of use of small organic compounds as ligands for modulation of PCSK9 biological activity. In particular, the invention provides compositions of small organic compounds that modulate circulating levels of low density lipoproteins by altering the conformation of the protein PCSK9. Binding these small organic compound ligands to PCSK9 alters the conformation of the protein, modifying the interaction between PCSK9 and an endogenous low density lipoprotein receptor, and can lead to reduced or increased levels of circulating LDL-cholesterol. High LDL-cholesterol levels are associated with increased risk for heart disease. Low LDL-cholesterol levels may be problematic in other conditions, such as liver dysfunction; thus, there is also utility for small organic compound ligands that can raise LDL levels.

This application is a continuation of U.S. application Ser. No.16/739,544, filed Jan. 10, 2020, which is a continuation of U.S.application Ser. No. 15/753,790, filed Feb. 20, 2018, now U.S. Pat. No.10,568,882, which is a U.S. National Stage Application under 35 U.S.C. §371 of International Application No. PCT/US2016/047816, filed Aug. 19,2016, which claims priority benefit of U.S. Provisional Application No.62/298,890, filed Feb. 23, 2016, and U.S. Provisional Application No.62/208,072, filed Aug. 21, 2015, the contents of each of which arehereby incorporated by reference in their entireties into the presentdisclosure.

FIELD OF INVENTION

This invention is related to the field of PCSK9 biology. In particular,the invention provides compositions and methods of use of small organiccompounds as ligands that directly bind with the PCSK9 protein, anddifferentially modify PCSK9 biological activity in cells. These changesmay include alteration in PCSK9 binding to, or dissociating from, LDLR,changes in LDLR number on the cell surface, or changes in the rate ofLDL internalization.

BACKGROUND

Elevated plasma levels of low density lipoprotein cholesterol (LDL-C)represent the greatest risk factor for the development of coronary heartdisease. Clearance of LDL-C from the plasma occurs primarily by theliver through the action of LDL receptors (LDLRs), which are cellsurface glycoproteins that bind to apolipoprotein B100 (apoB100) on LDLparticles with high affinity and mediate their endocytic uptake.Goldstein et al., Annu. Rev. Cell Biol. 1:1-39 (1985). Autosomaldominant hypercholesterolemia (ADH) is associated with mutations thatreduce plasma LDL clearance that are found in genes encoding the LDLR(familial hypercholesterolemia (FH)) or apoB100 (familial defectiveapoB100). Hobbs et al., Annu. Rev. Genet. 24, 133-170 (1990); andInnerarity et al., J. Lipid Res. 31:1337-1349 (1990), respectively.

The low density lipoprotein (LDL) receptor (LDLR) mediates efficientendocytosis of VLDL, VLDL remnants, and LDL. As part of the endocyticprocess, the LDLR releases lipoproteins into hepatic endosomes.

One approach to modulating LDL-cholesterol levels would be to identifysmall organic compounds that bind to PCSK9 and alter the kinetics of theinteraction between PCSK9 and the LDLR such that the rate of lipoproteinclearance by LDLR endocytosis is increased or decreased, as desired.

SUMMARY OF THE INVENTION

This invention is related to the field of PCSK9 biology and treatment ofhypercholesterolemia and hypocholesterolemia. In particular, theinvention provides compositions of ligands that bind and alter PCSK9biological conformation and methods that use these ligands to modifyPCSK9 activity to change circulating levels of low density lipoproteinin the blood. These ligands may be small organic compounds, and morepreferably small organic compounds less than 600 Da. Altering theconformation of PCSK9 can change the interactions between PCSK9 and anendogenous low density lipoprotein receptor, and can lead to reduced orincreased levels of circulating LDL-cholesterol. High LDL-cholesterollevels are associated with increased risk for heart disease. LowLDL-cholesterol levels may be problematic in other conditions, such asliver dysfunction; thus, there is also utility for ligands that canraise LDL levels.

Phenylpiperazine Scaffold:

In one embodiment, the present invention contemplates a phenylpiperazinesmall organic compound comprising of a phenylpiperazine scaffold ofFormula I:

wherein i) A is selected from the group consisting of —O—, —CO—, —CH₂—,—CF₂—, —SO₂—; ii) if A is —CH₂ or —CF₂ then B is absent, and if A is —O—then B is absent or —CH₂, and, if A is —CO or SO₂ then B is —NH—; iii)Ring C is a 5 or 6 membered saturated or unsaturated aryl, heteroaryl,carbocyclic or heterocyclic ring, wherein ring C may be optionally fusedwith R¹, R² to form a 5-6 membered aryl, heteroaryl, carbocyclic orheterocyclic ring; iv) E¹, E², and E³ are independently selected from C,CH, or N; v) if B is absent or —CH₂— then R¹ and R² are independentlyselected from the group consisting of H, lower alkyl, hydroxy, amino,aminoalkyl, hydroxyalkyl, haloalkyl, carboxy, —CONH₂, nitrile, —S-alkyl,—O-alkyl, acyl, and oxo; vi) if B is —NH— then R¹, R², and R³ areindependently selected from the group consisting of H, lower alkyl,hydroxy, amino, aminoalkyl, hydroxyalkyl, haloalkyl, carboxy, —CONH₂,—CON-alkyl, nitrile, —S-alkyl, —O-alkyl, acyl, oxo; or R¹ and R² withthe atoms attached thereof combine to form a 5-6 membered fusedsaturated or unsaturated ring D containing 0-3 heteroatoms where Ring Dmay further be substituted at positions at least two atoms away from thejuncture with Ring C; vi) R⁴ is selected from the group consisting of H,OH, halogen, and lower alkyl; vii) R⁵ is selected from the groupconsisting of H₂, and O; vii) Y is selected form the group consisting ofN, and CH; and viii) X¹⁻⁴ are independently selected from the groupconsisting of CH, C—R⁴, or N. Also provided are pharmaceuticallyacceptable salts, hydrates, solvates, tautomeric forms, andstereoisomers of these compounds. In one embodiment the compound isselected from the group comprising of:

-   N-phenyl-4-(piperazin-1-yl)benzenesulfonamide;-   N-isopropyl-4-((4-(piperazin-1-yl)phenyl)sulfonamido)benzamide;-   N-methyl-4-((4-(piperazin-1-yl)phenyl)sulfonamido)benzamide;-   N-cyclopentyl-4-(piperazin-1-yl)benzenesulfonamide;-   N-cyclohexyl-4-(piperazin-1-yl)benzenesulfonamide;-   N-(3,4-dimethylphenyl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(3,5-dimethylphenyl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(3,5-dichlorophenyl)-4-(piperazin-1-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;-   N-(naphthalen-2-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(cinnolin-7-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(isoquinolin-7-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(quinolin-6-yl)benzenesulfonamide;-   N-(cinnolin-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(quinoxalin-6-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(pyrido[2,3-b]pyrazin-7-yl)benzenesulfonamide;-   N-(1H-benzo[d][1,2,3]triazol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2-methylbenzo[b]thiophen-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(benzo[c][1,2,5]thiadiazol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(1H-indol-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(1H-indol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(benzo[d]isoxazol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2-aminoimidazo[1,2-a]pyrimidin-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2,3-dihydrobenzofuran-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2,3-dihydrobenzofuran-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-phenyl-4-(piperazin-1-yl)benzamide;-   N-(cinnolin-7-yl)-4-(piperazin-1-yl)benzamide;-   4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzamide;-   1-(4-(phenylsulfonyl)phenyl)piperazine;-   1-(4-((4-chlorophenyl)sulfonyl)phenyl)piperazine;-   4-((4-(piperazin-1-yl)phenyl)sulfonyl)phenol;-   1-methyl-4-((4-(piperazin-1-yl)phenyl)sulfonyl)pyridin-2(1H)-one;-   4-((4-(piperazin-1-yl)phenyl)sulfonyl)pyridin-2(1H)-one;-   1-(4-(difluoro(phenyl)methyl)phenyl)piperazine;-   1-(4-(difluoro(pyridin-3-yl)methyl)phenyl)piperazine;-   3-(difluoro(4-(piperazin-1-yl)phenyl)methyl)phenol;-   phenyl(4-(piperazin-1-yl)phenyl)methanone;-   1-(4-benzylphenyl)piperazine;-   1-(4-(benzyloxy)phenyl)piperazine;-   1-(4-phenoxyphenyl)piperazine;-   1-(4-(4-bromophenoxy)phenyl)piperazine;-   1-(4-(4-(methylthio)phenoxy)phenyl)piperazine;-   4-(4-(piperazin-1-yl)phenoxy)benzoic acid;-   4-(4-(piperazin-1-yl)phenoxy)benzamide;-   2-bromo-4-(4-(piperazin-1-yl)phenoxy)benzoic acid;-   5-(4-(piperazin-1-yl)phenoxy)picolinamide;-   1-(4-(3,4-difluorophenoxy)phenyl)piperazine;-   1-(4-(3,4-dimethylphenoxy)phenyl)piperazine;-   2-hydroxy-N-phenyl-4-(piperazin-1-yl)benzenesulfonamide;-   4-(2-oxopiperazin-1-yl)-N-phenylbenzenesulfonamide;-   2-fluoro-4-(2-oxopiperazin-1-yl)N-phenylbenzenesulfonamide;-   2-hydroxy-N-phenyl-4-(piperazin-1-yl)benzamide;-   1-(4-(difluoro(phenyl)methyl)-3-fluorophenyl)piperazin-2-one;-   2-(phenylsulfonyl)-5-(piperazin-1-yl)phenol;-   N-phenyl-4-(piperazin-1 -yl-2,2,6,6-d₄)benzenesulfonamide;-   4-(4-phenoxyphenyl)piperidine;-   4-(4-(cyclohexyloxy)phenyl)piperidine;-   4-(4-((tetrahydro-2H-pyran-4-yl)oxy)phenyl)piperidine;-   4-(4-(piperidin-4-yl)phenoxy)piperidine,-   1-(4-(cyclohexyloxy)phenyl)piperazine;-   1-(4-((tetrahydro-2H-pyran-4-yl)oxy)phenyl)piperazine;-   1-(4-(piperidin-4-yloxy)phenyl)piperazine;-   N-phenyl-4-(piperidin-4-yl)benzenesulfonamide;-   4-(piperidin-4-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;-   N-phenyl-4-(piperidin-4-yl)benzenesulfonamide;-   4-(piperidin-4-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;-   N-phenyl-4-(piperidin-4-yl)benzamide;-   4-(4-(difluoro(phenyl)methyl)phenyl)piperidine;-   phenyl(4-(piperidin-4-yl)phenyl)methanone;-   2-ethyl-N-phenyl-4-(piperazin-1-yl)benzamide;-   1-(4-phenoxyphenyl)piperazin-2-one;-   1-(4-(4-fluorobenzyl)phenyl)piperazine;-   6-(4-(piperazin-1-yl)phenoxy)pyrimidin-4(3H)-one;-   4-((4-(piperazin-1-yl)phenyl)sulfonyl)morpholine;-   1-(4-(pyrrolidin-1-ylsulfonyl)phenyl)piperazine;-   1-(2-fluoro-4-(phenylsulfonyl)phenyl)piperazine;-   4-(piperazin-1-yl)benzenesulfonamide;-   N-methyl-4-(piperazin-1-yl)benzenesulfonamide;-   N-cyclopropyl-4-(piperazin-1-yl)benzenesulfonamide;-   N-isopropyl-4-(piperazin-1-yl)benzenesulfonamide;-   N-((4-(piperazin-1-yl)phenyl)sulfonyl)acetamide;-   N-(4-(piperazin-1-yl)phenyl)methanesulfonamide;-   N-(4-(piperazin-1-yl)phenyl)ethanesulfonamide;-   N-(4-(piperazin-1-yl)phenyl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(pyridin-4-yl)benzenesulfonamide;-   N-(4-(piperazin-1-yl)phenyl)ethanesulfonamide;-   N-(4-(piperazin-1-yl)phenyl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(pyridin-3-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(pyridin-2-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(thiazol-2-yl)benzenesulfonamide:-   4-(piperazin-1-yl)-N-(quinolin-7-yl)benzenesulfonamide;-   N-phenyl-6-(piperazin-1-yl)pyridine-3-sulfonamide;-   N-phenyl-5-(piperazin-1-yl)pyridine-2-sulfonamide;-   N-(3,5-difluorophenyl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2,3-dihydro-1H-inden-2-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   3-fluoro-4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;-   6-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)pyridine-3-sulfonamide;-   5-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)pyridine-2-sulfonamide;-   N-phenyl-4-(piperidin-4-yl)benzenesulfonamide;-   4-(piperidin-4-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;-   1-(4-phenoxyphenyl)piperazine-2,2,3,3,5,5,6,6-d₈;-   and 1-(4-(4-fluorobenzyl)phenyl)piperazine-2,2,3,3,5,5,6,6-d₈;    or a pharmaceutical composition thereof.

In one embodiment, compounds of Formula I where one or more atoms aresubstituted with an isotope are also contemplated. For example,1-(4-phenoxyphenyl)piperazine is selectively substituted with deuteriumto form the compound 1-(4-phenoxyphenyl)piperazine-2,2,3,3,5,5,6,6-s.Another example, 1-(4-(4-fluorobenzyl)phenyl)piperazine is selectivelysubstituted with deuterium to form the compound1-(4-(4-fluorobenzyl)phenyl)piperazine-2,2,3,3,5,5,6,6-d. Other examplesof isotopes that can be incorporated into compounds of the disclosureinclude isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous,fluorine and chlorine, such as, but not limited to ²H (deuterium, d), 3H(tritium), ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F, ³¹P, ³²P, ³⁵S, ³⁶Cl and ¹²⁵I.

In one embodiment, the present invention contemplates a phenylpiperazinesmall organic compound comprising of a phenylpiperazine scaffold ofFormula II:

wherein i) A is selected from the group consisting of —O—, —CO—, —CH₂—,—CF₂—, —SO₂—; ii) if A is —CH₂ or —CF₂ then B is nothing, if A is —O—then B is nothing or —CH₂, if A is —CO or SO₂ then B is —NH—; iii) RingC is a 5 or 6 membered saturated or unsaturated aryl, heteroaryl,carbocyclic or heterocyclic ring. And ring C may be fused via R¹, R² toform a 5-6 membered aryl, heteroaryl, carbocyclic or heterocyclic ring;iv) E¹, E², and E³ are independently selected from C or N; v) if B isnothing or —CH₂— then R¹ and R² are independently selected from thegroup consisting of lower alkyl, hydroxy, amino, aminoalkyl,hydroxylalky, haloalkyl, carboxy, —CONH₂, nitrile, —S-alkyl, —O-alkyl,acyl, and oxo; vi) if B is —NH— then R¹, R², and R³ are independentlyselected from the group consisting of lower alkyl, hydroxy, amino,aminoalkyl, hydroxylalky, haloalkyl, carboxy, —CONH₂, —CON-alkyl,nitrile, —S-alkyl, —O-alkyl, acyl, oxo; and R¹ and R² may be joined toform a 5-6 membered fused saturated or unsaturated ring D containing 0-3heteroatoms where Ring D may further be substituted at positions atleast two atoms away from the juncture with Ring C; vi) R⁴ is selectedfrom the group consisting of H, OH, halogen, and lower alkyl; vii) R⁵ isselected from the group consisting of H₂, and O; vii) R⁶ is selectedform the group consisting of N, and CH.

In one embodiment, the present invention contemplates a phenylpiperazinesmall organic compound comprising of a phenylpiperazine scaffold ofFormula III:

wherein i) A is selected from the group consisting of —O—, —CO—, —CH₂—,—CF₂—, —SO₂—; ii) if A is —CH₂ or —CF₂ then B is nothing, if A is —O—then B is nothing or —CH₂, if A is —CO or SO₂ then B is —NH—; iii) RingC is a 5 or 6 membered aryl, heteroaryl, carbocyclic or heterocyclicring. And ring C may be fused via R¹, R² to form a 5-6 membered aryl,heteroaryl, carbocyclic or heterocyclic ring; iv) E¹, E², and E³ areindependently selected from C or N; v) if B is nothing or —CH₂— then R¹and R² are independently selected from the group consisting of loweralkyl, hydroxy, amino, aminoalkyl, hydroxylalky, haloalkyl, carboxy,—CONH₂, nitrile, —S-alkyl, —O— alkyl, acyl, and oxo; vi) if B is —NH—then R¹, R², and R³ are independently selected from the group consistingof lower alkyl, hydroxy, amino, aminoalkyl, hydroxylalky, haloalkyl,carboxy, —CONH₂, —CON-alkyl, nitrile, —S-alkyl, —O-alkyl, acyl, oxo; andR¹ and R² may be joined to form a 5-6 membered fused ring D containing0-3 heteroatoms where Ring D may further be substituted at positions atleast two atoms away from the juncture with Ring C; vi) R⁴ is selectedfrom the group consisting of H, OH, and halogen; vii) R³ is selectedfrom the group consisting of H₂, and O.

Compound Methods of Use:

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a PCSK9 protein, wherein said proteincomprises a binding site that induces allosteric modulation and a lowdensity lipoprotein receptor binding site; ii) a small organic compoundcapable of binding to said binding site, and selected from the groupconsisting of a compound of a phenylpiperazine scaffold of Formula I;iii) a plurality of hepatocyte cells comprising a low densitylipoprotein receptor and low density lipoproteins; b) binding said smallorganic compound to said binding site, wherein said small organiccompound induces a conformational shift of said protein; and c)modulating the rate of low density lipoprotein receptor internalizationby said plurality of hepatocytes by said conformational shift. In oneembodiment the present invention contemplates a method, wherein saidsmall organic compound induces a conformational change of the PCSK9protein, such that the rate of low density lipoprotein receptorinternalization by a plurality of hepatocytes is increased.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a PCSK9 protein, wherein said proteincomprises a binding site that induces allosteric modulation and a lowdensity lipoprotein receptor binding site; ii) a small organic compoundcapable of binding to said binding site, and selected from the groupconsisting of a compound of a phenylpiperazine scaffold of Formula I;iii) a plurality of hepatocyte cells comprising a low densitylipoprotein receptor and low density lipoproteins; b) binding said smallorganic compound to said binding site, wherein said small organiccompound induces a conformational shift of said protein; and c)modulating the affinity of said low density lipoprotein receptor bindingsite for said low density lipoprotein receptor by said conformationalshift.

In one embodiment, the small organic compound is an allosteric inhibitorligand wherein said modulating decreases the affinity of said lowdensity lipoprotein receptor binding site for said low densitylipoprotein receptor such that internalization of said low densitylipoprotein by said plurality of hepatocytes is increased. In oneembodiment, the conformational shift of said protein is selected fromthe group consisting of an induced fit shift and a biomechanical shift.In one embodiment the small organic compound is a small moleculecompound. In one embodiment, the small organic compound is selected fromthe group consisting of a phenylpiperazine scaffold of Formula I. In oneembodiment, the small organic compound is selected from the groupconsisting of a phenylpiperazine scaffold of Formula I, wherein one ormore atoms are substituted with an isotope are also contemplated. Alsoprovided are pharmaceutically acceptable salts, hydrates, solvates,tautomeric forms, and stereoisomers of these compounds selected from thegroup consisting of a phenylpiperazine scaffold of Formula I.

In one embodiment, the small organic compound is an allosteric inhibitorligand wherein said modulating decreases the affinity of said lowdensity lipoprotein receptor binding site for said low densitylipoprotein receptor such that internalization of said low densitylipoprotein by said plurality of hepatocytes is increased. In oneembodiment, the conformational shift of said protein is selected fromthe group consisting of an induced fit shift and a biomechanical shift.In one embodiment, the small organic compound is a small moleculecompound. In one embodiment, the small organic compound is selected fromthe group comprising of:

-   N-phenyl-4-(piperazin-1-yl)benzenesulfonamide;-   N-isopropyl-4-((4-(piperazin-1-yl)phenyl)sulfonamido)benzamide;-   N-methyl-4-((4-(piperazin-1-yl)phenyl)sulfonamido)benzamide;-   N-cyclopentyl-4-(piperazin-1-yl)benzenesulfonamide;-   N-cyclohexyl-4-(piperazin-1-yl)benzenesulfonamide;-   N-(3,4-dimethylphenyl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(3,5-dimethylphenyl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(3,5-dichlorophenyl)-4-(piperazin-1-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;-   N-(naphthalen-2-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(cinnolin-7-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(isoquinolin-7-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(quinolin-6-yl)benzenesulfonamide;-   N-(cinnolin-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(quinoxalin-6-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(pyrido[2,3-b]pyrazin-7-yl)benzenesulfonamide;-   N-(1H-benzo[d][1,2,3]triazol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2-methylbenzo[b]thiophen-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(benzo[c][1,2,5]thiadiazol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(1H-indol-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(1H-indol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(benzo[d]isoxazol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2-aminoimidazo[1,2-a]pyrimidin-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2,3-dihydrobenzofuran-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2,3-dihydrobenzofuran-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-phenyl-4-(piperazin-1-yl)benzamide;-   N-(cinnolin-7-yl)-4-(piperazin-1-yl)benzamide;-   4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzainide;-   1-(4-(phenylsulfonyl)phenyl)piperazine;-   1-(4-((4-chlorophenyl)sulfonyl)phenyl)piperazine;-   4-((4-(piperazin-1-yl)phenyl)sulfonyl)phenol;-   1-methyl-4-((4-(piperazin-1-yl)phenyl)sulfonyl)pyridin-2(1H)-one;-   4-((4-(piperazin-1-yl)phenyl)sulfonyl)pyridin-2(1H)-one;-   1-(4-(difluoro(phenyl)methyl)phenyl)piperazine;-   1-(4-(difluoro(pyridin-3-yl)methyl)phenyl)piperazine;-   3-(difluoro(4-(piperazin-1-yl)phenyl)methyl)phenol;-   phenyl(4-(piperazin-1-yl)phenyl)methanone,-   1-(4-benzylphenyl)piperazine;-   1-(4-(benzyloxy)phenyl)piperazine,-   1-(4-phenoxyphenyl)piperazine;-   1-(4-(4-bromophenoxy)phenyl)piperazine;-   1-(4-(4-(methylthio)phenoxy)phenyl)piperazine;-   4-(4-(piperazin-1-yl)phenoxy)benzoic acid;-   4-(4-(piperazin-1-yl)phenoxy)benzainide,-   2-bromo-4-(4-(piperazin-1-yl)phenoxy)benzoic acid;-   5-(4-(piperazin-1-yl)phenoxy)picolinamide,-   1-(4-(3,4-difluorophenoxy)phenyl)piperazine;-   1-(4-(3,4-dimethylphenoxy)phenyl)piperazine;-   2-hydroxy-N-phenyl-4-(piperazin-1-yl)benzenesulfonamide;-   4-(2-oxopiperazin-1-yl)-N-phenylbenzenesulfonamide;-   2-fluoro-4-(2-oxopiperazin-1-yl)-N-phenylbenzenesulfonamide;-   2-hydroxy-N-phenyl-4-(piperazin-1-yl)benzamide;-   1-(4-(difluoro(phenyl)methyl)-3-fluorophenyl)piperazin-2-one;-   2-(phenylsulfonyl)-5-(piperazin-1-yl)phenol;-   N-phenyl-4-(piperazin-1-yl-2,2,6,6-d4)benzenesulfonamide;-   4-(4-phenoxyphenyl)piperidine;-   4-(4-(cyclohexyloxy)phenyl)piperidine;-   4-(4-((tetrahydro-2H-pyran-4-yl)oxy)phenyl)piperidine;-   4-(4-(piperidin-4-yl)phenoxy)piperidine;-   1-(4-(cyclohexyloxy)phenyl)piperazine;-   1-(4-((tetrahydro-2H-pyran-4-yl)oxy)phenyl)piperazine;-   1-(4-(piperidin-4-yloxy)phenyl)piperazine;-   N-phenyl-4-(piperidin-4-yl)benzenesulfonamide;-   4-(piperidin-4-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;-   N-phenyl-4-(piperidin-4-yl)benzamide;-   4-(4-(difluoro(phenyl)methyl)phenyl)piperidine;-   phenyl(4-(piperidin-4-yl)phenyl)methanone;-   2-ethyl-N-phenyl-4-(piperazin-1-yl)benzamide;-   1-(4-phenoxyphenyl)piperazin-2-one;-   1-(4-(4-fluorobenzyl)phenyl)piperazine;-   6-(4-(piperazin-1-yl)phenoxy)pyrimidin-4(3H)-one;-   4-((4-(piperazin-1-yl)phenyl)sulfonyl)morpholine;-   1-(4-(pyrrolidin-1-ylsulfonyl)phenyl)piperazine;-   1-(2-fluoro-4-(phenylsulfonyl)phenyl)piperazine;-   4-(piperazin-1-yl)benzenesulfonamide;-   N-methyl-4-(piperazin-1-yl)benzenesulfonamide;-   N-cyclopropyl-4-(piperazin-1-yl)benzenesulfonamide;-   N-isopropyl-4-(piperazin-1-yl)benzenesulfonamide;-   N-((4-(piperazin-1-yl)phenyl)sulfonyl)acetamide;-   N-(4-(piperazin-1-yl)phenyl)methanesulfonamide;-   N-(4-(piperazin-1-yl)phenyl)ethanesulfonamide;-   N-(4-(piperazin-1-yl)phenyl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(pyridin-4-yl)benzenesulfonamide;-   N-(4-(piperazin-1-yl)phenyl)ethanesulfonamide;-   N-(4-(piperazin-1-yl)phenyl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(pyridin-3-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(pyridin-2-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(thiazol-2-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(quinolin-7-yl)benzenesulfonamide:-   N-phenyl-6-(piperazin-1-yl)pyridine-3-sulfonamide;-   N-phenyl-5-(piperazin-1-yl)pyridine-2-sulfonamide:-   N-(3,5-difluorophenyl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2,3-dihydro-1H-inden-2-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   3-fluoro-4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;-   6-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)pyridine-3-sulfonamide;-   5-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)pyridine-2-sulfonamide;-   N-phenyl-4-(piperidin-4-yl)benzenesulfonamide;-   4-(piperidin-4-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;-   1-(4-phenoxyphenyl)piperazine-2,2,3,3,5,5,6,6-d₈;-   and 1-(4-(4-fluorobenzyl)phenyl)piperazine-2,2,3,3,5,5,6,6-d₈;    or a pharmaceutical composition thereof.

In one embodiment, the present invention contemplates, a method,comprising: a) providing; i) a PCSK9 protein, wherein said proteincomprises a binding site that induces allosteric modulation and a lowdensity lipoprotein receptor binding site; ii) a small organic compoundcapable of binding said binding site, and selected from the groupconsisting of a compound of a phenylpiperazine scaffold of Formula I;iii) a plurality of hepatocyte cells comprising a population of lowdensity lipoprotein receptors; b) binding said small organic compound tosaid binding site, wherein said small organic compound induces aconformational shift of said protein; c) modulating said population ofsaid low density lipoprotein receptors by said conformational shift. Inone embodiment the present invention contemplates a method, wherein saidsmall organic compound induces a conformational change of the PCSK9protein, such that the population of low density lipoprotein receptorsis increased.

In one embodiment, the small organic compound is an allosteric inhibitorligand wherein said small molecule ligand induces a conformationalchange of the PCSK9 protein, such that the population of low densitylipoprotein receptors is increased. In one embodiment, the small organiccompound is an allosteric inhibitor ligand wherein said modulatingincreases said population of said low density lipoprotein receptorsmeasurable on the cell surface of said plurality of hepatocytes. In oneembodiment, the conformational shift of said protein is selected fromthe group consisting of an induced fit shift and a biomechanical shift.In one embodiment the small molecule compound is an organic chemicalcompound. In one embodiment, the small organic compound is selected fromthe group consisting of a phenylpiperazine scaffold of Formula I. In oneembodiment, the small organic compound is selected from the groupconsisting of a phenylpiperazine scaffold of Formula I, wherein one ormore atoms are substituted with an isotope are also contemplated. Alsoprovided are pharmaceutically acceptable salts, hydrates, solvates,tautomeric forms, and stereoisomers of these compounds selected from thegroup consisting of a phenylpiperazine scaffold of Formula I.

In one embodiment, the small organic compound is an allosteric inhibitorligand wherein said small molecule ligand induces a conformationalchange of the PCSK9 protein, such that the population of low densitylipoprotein receptors is increased. In one embodiment, the small organiccompound is an allosteric inhibitor ligand wherein said modulatingincreases said population of said low density lipoprotein receptorsmeasurable on the cell surface of said plurality of hepatocytes. In oneembodiment, the conformational shift of said protein is selected fromthe group consisting of an induced fit shift and a biomechanical shift.In one embodiment the small molecule compound is an organic chemicalcompound. In one embodiment, the small organic compound is selected fromthe group comprising of:

-   N-phenyl-4-(piperazin-1-yl)benzenesulfonamide;-   N-isopropyl-4-((4-(piperazin-1-yl)phenyl)sulfonamido)benzamide;-   N-methyl-4-((4-(piperazin-1-yl)phenyl)sulfonamido)benzamide;-   N-cyclopentyl-4-(piperazin-1-yl)benzenesulfonamide;-   N-cyclohexyl-4-(piperazin-1-yl)benzenesulfonamide;-   N-(3,4-dimethylphenyl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(3,5-dimethylphenyl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(3,5-dichlorophenyl)-4-(piperazin-1-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;-   N-(naphthalen-2-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(cinnolin-7-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(isoquinolin-7-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(quinolin-6-yl)benzenesulfonamide;-   N-(cinnolin-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(quinoxalin-6-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(pyrido[2,3-b]pyrazin-7-yl)benzenesulfonamide,-   N-(1H-benzo[d][l,2,3]triazol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2-methylbenzo[b]thiophen-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)-4-(piperazin-1-yl)benzenesulfomamide;-   N-(benzo[c][1,2,5]thiadiazol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(1H-indol-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(1H-indol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(benzo[d]isoxazol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2-aminoimidazo[1,2-a]pyrimidin-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2,3-dihydrobenzofuran-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2,3-dihydrobenzofuran-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2,2-difluorobenzo[d][3,3]dioxol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-phenyl-4-(piperazin-1-yl)benzarnide;-   N-(cinnolin-7-yl)-4-(piperazin-1-yl)benzamide;-   4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzamide;-   1-(4-(phenylsulfonyl)phenyl)piperazine;-   1-(4-((4-chlorophenyl)sulfonyl)phenyl)piperazine;-   4-((4-(piperazin-1-yl)phenyl)sulfonyl)phenol;-   1-methyl-4-((4-(piperazin-1-yl)phenyl)sulfonyl)pyridin-2(1H)-one;-   4-((4-(piperazin-1-yl)phenyl)sulfonyl)pyridin-2(1H)-one;-   4-((4-(piperazin-1-yl)phenyl)sulfonyl)pyridin-2(1H)-one;-   1-(4-(difluoro(phenyl)methyl)phenyl)piperazine;-   1-(4-(difluoro(pyridin-3-yl)methyl)phenyl)piperazine;-   3-(difluoro(4-(piperazin-1-yl)phenyl)methyl)phenol;-   phenyl(4-(piperazin-1-yl)phenyl)methanone;-   1-(4-benzylphenyl)piperazine,-   1-(4-(benzyloxy)phenyl)piperazine;-   1-(4-phenoxyphenyl)piperazine;-   1-(4-(4-bromophenoxy)phenyl)piperazine;-   1-(4-(4-(methylthio)phenoxy)phenyl)piperazine;-   4-(4-(piperazin-1-yl)phenoxy)benzoic acid;-   4-(4-(piperazin-1-yl)phenoxy)benzamide;-   2-bromo-4-(4-(piperazin-1-yl)phenoxy)benzoic acid;-   5-(4-(piperazin-1-yl)phenoxy)picolinamide;-   1-(4-(3,4-difluorophenoxy)phenyl)piperazine;-   1-(4-(3,4-di methylphenoxy)phenyl)piperazine;-   2-hydroxy-N-phenyl-4-(piperazin-1-yl)benzenesulfonamide;-   4-(2-oxopiperazin-1-yl)-N-phenylbenzenesulfonamide;-   2-fluoro-4-(2-oxopiperazin-1-yl)-N-phenylbenzenesulfonamide;-   2-hydroxy-N-phenyl-4-(piperazin-1-yl)benzamide;-   1-(4-(difluoro(phenyl)methyl)-3-fluorophenyl)piperazin-2-one;-   2-(phenylsulfonyl)-5-(piperazin-1-yl)phenol;-   N-phenyl-4-(piperazin-1-yl-2,2,6,6-d4)benzenesulfonamide;-   4-(4-phenoxyphenyl)piperidine;-   4-(4-(cyclohexyloxy)phenyl)piperidine,-   4-(4-((tetrahydro-2H-pyran-4-yl)oxy)phenyl)piperidine;-   4-(4-(piperidin-4-yl)phenoxy)piperidine;-   1-(4-(cyclohexyloxy)phenyl)piperazine;-   1-(4-((tetrahydro-2H-pyran-4-yl)oxy)phenyl)piperazine;-   1-(4-(piperidin-4-yloxy)phenyl)piperazine,-   N-phenyl-4-(piperidin-4-yl)benzenesulfonamide;-   4-(piperidin-4-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide,-   N-phenyl-4-(piperidin-4-yl)benzamide;-   4-(4-(difluoro(phenyl)methyl)phenyl)piperidine;-   phenyl(4-(piperidin-4-yl)phenyl)methanone;-   2-ethyl-N-phenyl-4-(piperazin-1-yl)benzamide;-   1-(4-phenoxyphenyl)piperazin-2-one;-   1-(4-(4-fluorobenzyl)phenyl)piperazine,-   6-(4-(piperazin-1-yl)phenoxy)pyrimidin-4(3H)-one;-   4-((4-(piperazin-1-yl)phenyl)sulfonyl)morpholine;-   1-(4-(pyrrolidin-1-ylsulfonyl)phenyl)piperazine,-   1-(2-fluoro-4-(phenylsulfonyl)phenyl)piperazine;-   4-(piperazin-3-yl)benzenesulfonamide;-   N-methyl-4-(piperazin-1-yl)benzenesulfonamide;-   N-cyclopropyi-4-(piperazin-1-yl)benzenesulfonamide,-   N-isopropyl-4-(piperazin-1-yl)benzenesulfonamide;-   N-((4-(piperazin-1-yl)phenyl)sulfonyl)acetamide;-   N-(4-(piperazin-1-yl)phenyl)methanesulfonamide;-   N-(4-(piperazin-1-yl)phenyl)ethanesu1fonamide;-   N-(4-(piperazin-1-yl)phenyl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(pyridin-4-yl)benzenesulfonamide,-   N-(4-(piperazin-1-yl)phenyl)ethanesulfonamide;-   N-(4-(piperazin-1-yl)phenyl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-(pyridin-3-yl)benzenesulfonamide;-   4-(piperazin-1-yl)-N-pyridin-2-yl)benzenesulfonamide,-   4-(piperazin-1-yl)-N-(thiazol-2-yl)benzenesulfonamide;-   4-(piperazin-l-yl)-N-(quino3in-7-yl)benzenesulfonamide;-   N-phenyl-6-(piperazin-1-yl )pyridine-3-sulfonamide;-   N-phenyl-5-(piperazin-1-yl)pyridine-2-sulfonamide;-   N-(3,5-difluorophenyl)-4-(piperazin-1-yl)benzenesulfonamide;-   N-(2,3-dihydro-1H-inden-2-yl)-4-(piperazin-1-yl)benzenesulfonamide;-   3-fluoro-4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;-   6-(piperazin-1l-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)pyridine-3-sulfonamide;-   5-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)pyridine-2-sulfonamide,-   N-phenyl-4-(piperidin-4-yl)benzenesulfonamide,-   4-(piperidin-4-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;-   1-(4-phenoxyphenyl)piperazine-2,2,3,3,5,5,6,6-d8;-   and 1-(4-(4-fluorobenzyl)phenyl)piperazine-2,2,3,3,5,5,6,6-d8;    or a

In one embodiment, the present invention contemplates a compoundincluding, but not limited to, a phenylpiperazine scaffold of Formula I.In one embodiment, the PCSK9 binding small organic ligand is selectedfrom the group consisting of: a phenylpiperazine scaffold of Formula I.In one embodiment, the compound is formulated as a pharmaceuticalcomposition. In one embodiment, the pharmaceutical composition furthercomprises a pharmaceutical drug. In one embodiment, the pharmaceuticaldrug is selected from the group consisting of a statin, a cardiovasculardrug, a metabolic drug, and an antihypertensive drug. In one embodiment,the pharmaceutical drug is selected from the group consisting ofezetimibe, amlodipine besylate, sitagliptin, metformin, atorvastatin,rosuvastatin and simvastatin. In one embodiment, the pharmaceuticalcomposition is formulated as selected from the group consisting of atablet, a liquid, a gel, a capsule, a sachet, a microparticle, aliposome, a nanoparticle, a salt, a transdermal patch, an ointment, alotion, a cream, a gel, a drop, a strip, a suppository, a spray and apowder.

In one embodiment, the present invention contemplates a compositioncomprising a PCSK9 allosteric small organic compound selected from thegroup consisting of a phenylpiperazine scaffold of Formula I as a smallmolecule ligand. In one embodiment, the composition is a pharmaceuticalcomposition. In one embodiment, the pharmaceutical composition comprisesan effective dose of said ligand. In one embodiment, the pharmaceuticalcomposition comprises salts. In one embodiment, the pharmaceuticalcomposition is formulated for oral administration.

In one embodiment, the present invention contemplates a method,comprising: a) administering to a subject a small organic compoundselected from the group consisting of a phenylpiperazine scaffold ofFormula I, which binds PCSK9 and is an allosteric modulator of theprotein, wherein said subject has at least one symptom of acardiovascular disease; and b) reducing said at least one symptom ofcardiovascular disease by said PCSK9 allosteric modulator small moleculecompound administration. In one embodiment, said at least one symptom isreduced between 10%-85%. In one embodiment, said at least one symptom isreduced between 20%-65%. In one embodiment, said at least one symptom isreduced between 30%-55%. In one embodiment, the cardiovascular diseasecomprises a coronary disease. In one embodiment, the cardiovasculardisease comprises hypertension. In one embodiment, the cardiovasculardisease comprises hypercholesterolemia. In one embodiment, thecardiovascular disease comprises atherosclerosis. In one embodiment, theat least one symptom comprises reduced circulating high densitylipoprotein. In one embodiment, the at least one symptom compriseselevated circulating cholesterol. In one embodiment, the at least onesymptom comprises elevated circulating low density lipoprotein. In oneembodiment, the at least one symptom comprises high blood pressure. Inone embodiment, the administering comprises an effective dose of saidPCSK9 allosteric modulator small organic compound. In one embodiment,said administering further comprises a delivery system selected from thegroup including, but not limited to, liposomes, microparticles andnanoparticles. In one embodiment, the effective dose comprises apharmaceutical composition. In one embodiment, the pharmaceuticalcomposition comprises salts. In one embodiment, the pharmaceuticalcomposition is formulated for oral administration. In one embodiment theallosteric modulator small organic compound is an organic chemicalcompound small molecule. In one embodiment, the small organic compoundis selected from the group consisting of a phenylpiperazine scaffold ofFormula I.

In one embodiment, the present invention contemplates a method,comprising: a) administering a PCSK9 allosteric small organic compoundselected from the group consisting of a phenylpiperazine scaffold ofFormula I to a subject, wherein said subject has at least one symptom ofa liver disease; and b) reducing said at least one symptom of liverdisease by said PCSK9 allosteric small molecule administration. In oneembodiment, the at least one symptom comprises elevated low densitylipoprotein receptor density. In one embodiment the at least one symptomcomprises reduced low density lipoprotein receptor density. In oneembodiment, said at least one symptom is reduced between 10%-85%. In oneembodiment, said at least one symptom is reduced between 20%-65%. In oneembodiment, said at least one symptom is reduced between 30%-55%. In oneembodiment, the PCSK9 allosteric small organic compound comprises aPCSK9 allosteric inhibitor compound. In one embodiment, theadministering comprises an effective dose of said PCSK9 allostericinhibitor compound. In one embodiment, said administering furthercomprises a delivery system selected from the group including, but notlimited to, liposomes, microparticles and nanoparticles. In oneembodiment, the effective dose comprises a pharmaceutical composition.In one embodiment, the pharmaceutical composition comprises salts. Inone embodiment, the pharmaceutical composition is formulated for oraladministration. In one embodiment the small organic compound is anorganic chemical compound small molecule. In one embodiment, the smallorganic compound is selected from the group consisting of aphenylpiperazine scaffold of Formula I

In one embodiment, the present invention contemplates a method,comprising: a) administering a PCSK9 allosteric small organic compoundselected from the group consisting of a phenylpiperazine scaffold ofFormula I to a subject, wherein said subject has at elevated PCSK9protein levels in the blood; and b) reducing said at least one symptomof elevated PCSK9 by said PCSK9 allosteric small molecule compoundadministration. In one embodiment, the at least one symptom comprisesreduced low density lipoprotein receptor density. In one embodiment,said at least one symptom is reduced between 10%-85%. In one embodiment,said at least one symptom is reduced between 20%-65%. In one embodiment,said at least one symptom is reduced between 30%-55%. In one embodiment,the PCSK9 allosteric small molecule compound comprises a PCSK9allosteric inhibitor compound. In one embodiment, the administeringcomprises an effective dose of said PCSK9 allosteric small moleculecompound. In one embodiment, said administering further comprises adelivery system selected from the group including, but not limited to,liposomes, microparticles and nanoparticles. In one embodiment, theeffective dose comprises a pharmaceutical composition. In oneembodiment, the pharmaceutical composition comprises salts. In oneembodiment, the pharmaceutical composition is formulated for oraladministration. In one embodiment the small molecule compound is anorganic chemical compound. In one embodiment, the small organic compoundis selected from the group consisting of a phenylpiperazine scaffold ofFormula I.

In one embodiment, the present invention contemplates a method,comprising: a) administering a PCSK9 allosteric small organic compoundselected from the group consisting of a phenylpiperazine scaffold ofFormula I to a subject, wherein said subject has at below-average PCSK9protein levels in the blood; and b) reducing said at least one symptomof elevated PCSK9 by said PCSK9 allosteric small molecule compoundadministration. In one embodiment, the at least one symptom compriseselevated low density lipoprotein receptor density. In one embodiment,said at least one symptom is reduced between 10%-85%. In one embodiment,said at least one symptom is reduced between 20%-65%. In one embodiment,said at least one symptom is reduced between 30%-55%. In one embodiment,the PCSK9 allosteric small molecule compound comprises a PCSK9allosteric activator compound. In one embodiment, the administeringcomprises an effective dose of said PCSK9 allosteric small moleculecompound. In one embodiment, said administering further comprises adelivery system selected from the group including, but not limited to,liposomes, microparticles and nanoparticles. In one embodiment, theeffective dose comprises a pharmaceutical composition. In oneembodiment, the pharmaceutical composition comprises salts. In oneembodiment, the pharmaceutical composition is formulated for oraladministration. In one embodiment the small molecule compound is anorganic chemical compound. In one embodiment, the small organic compoundis selected from the group consisting of a phenylpiperazine scaffold ofFormula I.

In one embodiment, the present invention further contemplates a kitcomprising (a) a first container comprising a pharmaceutical compositionof a small organic compound of Formula I; and (b) instructions for theuse thereof for treatment of hypercholesterolemia. In one embodiment,the present invention further contemplates a kit comprising (a) firstcontainer comprising a pharmaceutical composition of a small organiccompound of Formula I; and (b) instructions for the use thereof forinhibition of PCSK9 protein biological activity.

In one embodiment, the present invention further contemplates a smallorganic compound of Formula I for use in treating hypercholesterolemia.In one embodiment, the present invention further contemplates a smallorganic compound of Formula I for use in lowering serum LDL levels.

In one embodiment, the present invention further contemplates a methodfor reducing LDL levels in a mammal comprising administering apharmaceutical composition of a small organic compound of Formula I tothe mammal. In one embodiment, the present invention furthercontemplates a method for reducing LDL levels in a patient comprisingadministering a pharmaceutical composition of a small organic compoundof Formula I to the patient.

In one embodiment, the present invention further contemplates a methodfor treating hypercholesterolemia in a mammal comprising administering apharmaceutical composition of a small organic compound of Formula I tothe mammal. In one embodiment, the present invention furthercontemplates a method for treating hypercholesterolemia in a patientcomprising administering a pharmaceutical composition of a small organiccompound of Formula I to the patient.

In one embodiment, the present invention further contemplates a methodfor reducing cholesterol levels in a patient in need thereof, whereinsaid method comprises identifying a patient with elevated serumcholesterol levels, and administering a pharmaceutical composition of asmall organic compound of Formula I to the patient. In one embodiment,the present invention further contemplates a method for reducing LDLlevels in a patient in need thereof, wherein said method comprisesidentifying a patient with elevated serum LDL levels, and administeringa pharmaceutical composition of a small organic compound of Formula I tothe patient. In one embodiment, the present invention furthercontemplates a method for treating hypercholesterolemia in a patient inneed thereof, wherein said method comprises identifying a patient withelevated serum LDL levels, and administering a pharmaceuticalcomposition of a small organic compound of Formula I to the patient.

Definitions

The following abbreviations are used throughout the specification:

Bn: benzyl

Bz: benzoyl

Ac: acetyl

Boc: tert-butoxycarbonyl

Fmoc: 9-fluorenylmethoxycarbonyl

Cbz: benzyloxycarbonyl

TFA: trifluoroacetic acid

NMP: N-methylpyrrolidone

PyBOP:(benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate

NMM: N-methyl morpholine

HPLC: high pressure liquid chromatography

THF: tetrahydrofuran

DMSO: dimethylsulfoxide

DMF: N,N-dimethylformamide

TMS-Br: trimethylsilyl bromide

Tf: trifluoromethylsulfonyl

HATU:2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate

DIPEA: diisopropylethylamine

DCM: dichloromethane

The terms “Pharmaceutically acceptable” or “physiologically acceptable”refer to compounds, salts, compositions, dosage forms and othermaterials which are useful in preparing a pharmaceutical compositionthat is suitable for veterinary or human pharmaceutical use.

The term “pharmaceutically acceptable salt” of a given compound refersto salts that retain the biological effectiveness and properties of thegiven compound, and which are not biologically or otherwise undesirable.“Pharmaceutically acceptable salts” or “physiologically acceptablesalts” include, for example, salts with inorganic acids and salts withan organic acid. In addition, if the compounds described herein areobtained as an acid addition salt, the free base can be obtained bybasifying a solution of the acid salt. Conversely, if the product is afree base, an addition salt, particularly a pharmaceutically acceptableaddition salt, may be produced by dissolving the free base in a suitableorganic solvent and treating the solution with an acid, in accordancewith conventional procedures for preparing acid addition salts from basecompounds. Those skilled in the art will recognize various syntheticmethodologies that may be used to prepare nontoxic pharmaceuticallyacceptable addition salts. Pharmaceutically acceptable acid additionsalts may be prepared from inorganic and organic acids. Salts derivedfrom inorganic acids include hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derivedfrom organic acids include acetic acid, propionic acid, glycolic acid,pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid,maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluene-sulfonic acid, salicylic acid, and the like. Likewise,pharmaceutically acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases include,by way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines, such asalkyl amines (i.e., NH₂(alkyl)), dialkyl amines (i.e., HN(alkyl)₂),trialkyl amines (i.e., N(alkyl)₃), substituted alkyl amines (i.e.,NH₂(substituted alkyl)), di(substituted alkyl) amines (i.e.,HN(substituted alkyl)₂), tri(substituted alkyl) amines (i.e.,N(substituted alkyl)₃), alkenyl amines (i.e., NH₂(alkenyl)), dialkenylamines (i.e., HN(alkenyl)₂), trialkenyl amines (i.e., N(alkenyl)₃),substituted alkenyl amines (i.e., NH₂(substituted alkenyl)),di(substituted alkenyl) amines (i.e., HN(substituted alkenyl)₂),tri(substituted alkenyl) amines (i.e., N(substituted alkenyl)₃, mono-,di- or tri-cycloalkyl amines (i.e., NH₂(cycloalkyl), HN(cycloalkyl)₂,N(cycloalkyl)₃), mono-, di- or tri-arylamines (i.e., NH₂(aryl),HN(aryl)₂, N(aryl)₃), or mixed amines, etc. Specific examples ofsuitable amines include, by way of example only, isopropylamine,trimethyl amine, diethyl amine, tri(iso-propyl) amine, tri(n-propyl)amine, ethanolamine, 2-dimethylaminoethanol, piperazine, piperidine,morpholine, N-ethylpiperidine, and the like.

The term “hydrate” refers to the complex formed by the combining of acompound of Formula I and water.

The term “solvate” refers to an association or complex of one or moresolvent molecules and a compound of the disclosure. Examples of solventsthat form solvates include, but are not limited to, water, isopropanol,ethanol, methanol, dimethylsulfoxide, ethylacetate, acetic acid, andethanolamine.

The term “small molecule compound” as used herein, refers to anexogenously synthesized organic chemical compound of less than 1,000 Da.

The term “small molecule ligand” as used herein, refers to a smallmolecule compound that is bound by another naturally occurringbiomolecule to form a complex.

The term “small organic compound” as used herein, refers to a smallmolecule compound, and is synonymous with “organic compound” and “smallmolecule”.

The term “small organic compound ligand” as used herein refers to asmall organic compound that is bound by another naturally occurringbiomolecule to form a complex.

The term “conformation” as used herein, refers to a three-dimensionalstereochemical configuration of any compound and/or molecule. Forexample, any specific conformation results from a thermodynamic balancebetween steric interactions, hydrophobic interactions, hydrogen bonding,electrochemical bonding and/or salt bridge interactions in a protein.

The term “LDL-R” and “LDLR” as used herein, refers to an abbreviationfor the low density lipoprotein receptor. The abbreviation may be inreference to the entire LDL-R receptor protein or any portion thereof.LDL-Rs reside on a cell surface and can bind to low density lipoproteinssuch that the LDL-R/LDL complex become internalized within a cell (i.e.,for example, a hepatocyte), wherein the LDL is released and the LDL-R isrecycled back to the cell surface.

The term, “binding interface” as used herein, refers to any collectionof attractive interactions (i.e., for example, hydrogen bonding,electrostatic interactions, hydrophobic interactions, etc) between thefunctional groups (i.e., for example, hydroxyl, amide, amine, carboxyl,amidine, guanidine, hydrocarbon, sulfonyl etc.) of at least twodifferent molecules. The collection of attractive forces forms a stablemolecular plane thereby forming a ‘binding interface’ between the atleast two molecules.

The term “induced fit” as used herein, refers to any acceptance of asmall molecule compound requiring a change in the receiving molecule'sconformation. Such a conformation may be facilitated by atranslational/rotational movement of amino acid side chains and flexibleloops, thereby rearranging the electrostatic and/or hydrophobic fields.

The term “complex” or “composition” as used herein, refers to anychemical association of two or more ions or molecules joined usually byweak electrostatic bonds rather than by covalent bonds. For example, acomplex or composition may be formed between a small molecule compoundas described herein and a PCSK9 amino acid sequence, thereby creating asmall molecule compound:PCSK9 amino acid sequence complex orcomposition. Optionally, such complexes or compositions may alsoinclude, but are not limited to, an LDLR amino acid sequence or anyportion thereof, including but not limited to the EGFA region.

The term “hydrogen bond” as used herein, an electrostatic attractionbetween a hydrogen atom in one polar molecule (as of water) and a smallelectronegative atom (as of oxygen, nitrogen, or fluorine) in usuallyanother molecule of the same or a different polar substance.

The term “salt bridge” as used herein, refers to any interaction or acombinations of interactions, such as hydrogen bonding and/orelectrostatic interactions, which align cationic and anionic chemicalstructures in such a way that the charged moieties overlap.

The term “interaction” as used herein, refers to any effect that onemolecule and/or functional group may have on another molecule and/orfunctional group. Such effects may include, but are not limited to,steric (i.e., for example, physical), electrostatic (i.e., for example,electrical attraction or repulsion), electromagnetic, hydrophilic, orhydrophobic effects.

The term “overlap” as used herein, refers to any positioning ofmolecules in such a way that the electronic structure of one molecule ison top of, and extending past the border of another molecule, or bepositioned in this way.

The term “hypercholesterolemia” as used herein, refers to any medicalcondition wherein blood cholesterol levels are elevated above theclinically recommended levels. For example, if cholesterol is measuredusing low density lipoproteins (LDLs), hypercholesterolemia may exist ifthe measured LDL levels are above, for example, approximately 75 mg/dl.Alternatively, if cholesterol is measured using free plasma cholesterol,hypercholesterolemia may exist if the measured free cholesterol levelsare above, for example, approximately 200-220 mg/dl.

The term “hypocholesterolemia” as used herein, refers to any medicalcondition wherein blood cholesterol levels are below clinicallyrecommended levels. For example, if total cholesterol or LDL-C levelsare measured as below the 5^(th) percentile of the general populationafter adjustment for gender, race, and age.

The term “symptom”, as used herein, refers to any subjective orobjective evidence of disease or physical disturbance observed by thepatient. For example, subjective evidence is usually based upon patientself-reporting and may include, but is not limited to, pain, headache,visual disturbances, nausea and/or vomiting.

Alternatively, objective evidence is usually a result of medical testingincluding, but not limited to, body temperature, complete blood count,lipid panels, thyroid panels, blood pressure, heart rate,electrocardiogram, tissue and/or body imaging scans.

The term “disease” and/or “disorder”, as used herein, refers to anyimpairment of the normal state of the living animal or plant body or oneof its parts that interrupts or modifies the performance of the vitalfunctions. Typically manifested by distinguishing signs and symptoms, itis usually a response to: i) environmental factors (as malnutrition,industrial hazards, or climate); ii) specific infective agents (asworms, bacteria, or viruses); iii) inherent defects of the organism (asgenetic anomalies); and/or iv) combinations of these factors

The term “affinity” as used herein, refers to the measure of thethermodynamic tendency of two or more molecules to assemble to form amulti-part complex and to remain assembled in said complex. For example,a small molecule ligand has a high affinity for PCSK9 and isthermodynamically favored to form a complex. It is understood that achange in conditions (e.g., pH during the receptor internalizationprocess) may reduce the affinity of the molecules such that theydissociate, or separate, from one another. For example, pH changes canresult in a decrease in the LDL affinity for LDLR and subsequentdissociation of that complex.

The term “derived from” as used herein, refers to the source of acompound. In one respect, a compound may be derived from an organism orparticular species. In another respect, a compound may be derived from alarger complex. In another respect, a compound may be derived bychemical modification of part or all of a larger complex.

The term “protein” as used herein, refers to any of numerous naturallyoccurring extremely complex substances (as an enzyme or antibody) thatconsist of amino acid residues joined by peptide bonds, contain theelements carbon, hydrogen, nitrogen, oxygen, usually sulfur, and forminga contiguous protein backbone. In general, a protein comprises aminoacids having an order of magnitude within the hundreds.

The term “peptide” as used herein, refers to any of various amides thatare derived from three or more amino acids by combination of the aminogroup of one acid with the carboxyl group of another and are usuallyobtained by partial hydrolysis of proteins. In general, a peptidecomprises amino acids having an order of magnitude within the tens orsmaller.

The term, “purified” or “isolated”, as used herein, may refer to acomposition that has been subjected to treatment (i.e., for example,fractionation) to remove various other components, and which compositionsubstantially retains its expressed biological activity.

As used herein, the term “substantially purified” refers to molecules,such as small molecule compound, that are removed from their normalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and more preferably 90% free from other componentswith which they are normally associated.

The term “purified to homogeneity” is used to include compositions thathave been purified to “apparent homogeneity” such that there is singlesmall molecule compound species (i.e., for example, based upon SDS-PAGEor HPLC analysis). A purified composition is not intended to mean thatall trace impurities have been removed.

The term “biocompatible”, as used herein, refers to any material doesnot elicit a substantial detrimental response in the host. There isalways concern, when a foreign object is introduced into a living body,that the object will induce an immune reaction, such as an inflammatoryresponse that will have negative effects on the host. In the context ofthis invention, biocompatibility is evaluated according to theapplication for which it was designed: for example; a bandage isregarded a biocompatible with the skin, whereas an implanted medicaldevice is regarded as biocompatible with the internal tissues of thebody. Preferably, biocompatible materials include, but are not limitedto, biodegradable and biostable materials.

The terms “amino acid sequence” and “polypeptide sequence” as usedherein, are interchangeable and to refer to a contiguous sequence ofmultiple amino acids.

The term “derivative” as used herein, refers to any chemicalmodification of a small molecule compound. Examples of suchmodifications would include, but are not limited to, replacement of ahydrogen by an alkyl, aryl, hydroxyl, sulfhydryl, sulfoxyl, sulfonyl,acyl, phosphoryl, alkoxyl, amino or amino heterocyclic group. Otherpossible chemical modification might include, but are not limited to,C-terminal amides, and acyl or sulfonyl N-terminal modifications.

The term “bind” as used herein, includes any physical attachment orclose association, which may be permanent or temporary. Generally, aninteraction of hydrogen bonding, hydrophobic forces, van der Waalsforces, covalent and ionic bonding etc., facilitates physical attachmentbetween the molecule of interest and the analyte/target beingmeasuring/affected. The “binding” interaction may be brief as in thesituation where binding causes a chemical reaction to occur. That istypical when the binding component is an enzyme and the analyte/targetis a substrate for the enzyme. Reactions resulting from contact betweenthe binding agent and the analyte/target are also within the definitionof binding for the purposes of the present invention.

Chemical Terminology:

-   -   Alkyl: a chain consisting of only carbon and hydrogen atoms such        that each carbon atom directly connects to exactly 4 different        atoms, using only single bonds.    -   Lower alkyl: an alkyl chain containing 1-6 carbon atoms.    -   Branched alkyl: an alkyl chain containing one or more carbon        atoms which are directly connected to more than 2 other carbon        atoms without creating a ring of carbon atoms.    -   Hydroxyalkyl: an alkyl chain where at least one carbon atom is        bonded to a hydroxyl, that is, —OH.    -   Cycloalkyl: an alkyl chain forming a ring. Examples would        include—cyclopropyl or—cyclohexyl.    -   Heterocycle: a chain of atoms forming a ring and containing one        or more “heteroatoms”; that is, atoms other than C or H able to        form stable covalent bonds, such as N, O, or S. In this context,        “heterocyle” will imply a non-aromatic ring. Examples include a        tetrahydrofuran ring, with 4 carbon atoms and one oxygen, or a        morpholine, with 4 carbon atoms and one nitrogen and one oxygen        arranged such that the N and O are 1,4 to one another.    -   Aromatic ring: a ring of atoms containing alternating single and        double “pi” bonds such that the number pi electrons (typically 2        per double bonds for stable compounds) is an even number but not        a multiple of four.    -   Acyl: a carbonyl containing radical: —CO—R. In this document,        R=affords a typical peptide modifying group, such as: —CH₃        (acetyl), —CH(CH₂)₂ (isobutyryl).    -   Benzoyl: a carbonyl containing radical: —CO-Ph, where Ph=phenyl.    -   Sulfonyl: a sulfonyl containing radical: —SO₂—R.    -   Carbamoyl: a radical: —CONR₁R₂    -   Alkoxy: an alkyl chain containing one or more ether (—O—)        linkages, such as: —CH₂CH₂OCH₃.    -   Aryl: phenyl or substituted phenyl    -   Heteroaryl: a 5 or 6 membered aromatic heterocycle    -   Fused heterocyle: a ring system, such as indole, containing two        or more fused rings, of which at least one is a heterocycle. The        rings need not be aromatic: indoline has an aromatic ring fused        to a non-aromatic ring.    -   Negatively charged polar group: A polar group carrying a        negative charge at physiologic pH.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows exemplary data of % PCSK9 modulation in HepG2 cells in aDiI-LDL Uptake Assay. The cells were incubated in a 96-well plate for atotal of 16 h in the absence or presence of recombinant PCSK9 (finalconcentration 2.5 ug/ml) protein alone or recombinant PCSK9 proteinpre-mixed with indicated concentration of experimental compounds (Cmpd#). After 16 h, DiI-LDL (final concentration 5 ug/ml) was added to theincubation mixtures. After 4 h, cells are stained with Hoechst 33342 for30 minutes, then rinsed with phosphate buffered saline, and then a finalvolume of 100 ul phosphate buffered saline was added to each well. Thefluorescence was measured (DiI: excitation at 550 nm and emission at 590nm; Hoechst: excitation at 355 nm, emission at 460 nm). The percentrecombinant PCSK9 inhibition was calculated as [‘Compound Dose DiIFluorescence’−‘2.5 ug/ml PCSK9-treated DiI Fluorescence’]/[‘No PCSK9 DiIFluorescence’−‘2.5 ug/ml PCSK9-treated DiI Fluorescence’]×100%.

FIG. 2 shows exemplary data of % PCSK9 inhibition in HuH7 cells in anLDLR FACS Assay. The cells were incubated in 24-well plates for a totalof 6 h in the absence or presence of recombinant PCSK9 protein (finalconcentration of 5.0 ug/ml), or in the presence of PCSK9 protein mixedwith indicated concentration of experimental compound (Cmpd #). After 6hours, the cells were released from the plate, stained with anti-LDLRantibody, then rinsed, counterstained with DAPI, and then measured byfluorescence activated cell sorting (FACS). The percent recombinantPCSK9 inhibition was calculated as [‘Compound Dose LDLRFluorescence’−‘5.0 ug/ml PCSK9-treated LDLR Fluorescence’]/[‘No PCSK9Basal LDLR Fluorescence’−‘5.0 ug/ml PCSK9-treated LDLRFluorescence’]×100%.

DETAILED DESCRIPTION OF THE INVENTION

This invention is related to the field of PCSK9 biology and use ofcompounds for treatment including conditions of hypercholesterolemia andhypocholesterolemia. In particular, the invention provides compositionsof ligands that bind and alter PCSK9 biological conformation andactivity. These ligands are small molecule chemical compounds, and morepreferably small molecule compounds less than 800 Da. Altering theconformation of PCSK9 can change the interactions between PCSK9 and anendogenous low density lipoprotein receptor, and can lead to reduced orincreased levels of circulating LDL-cholesterol. High LDL-cholesterollevels are associated with increased risk for heart disease. LowLDL-cholesterol levels may be problematic in other conditions, such asliver dysfunction; thus, there is also utility for ligands that canraise LDL levels.

I. Physiological Role of Native PCSK9

Proprotein convertase subtilisin/kexin type 9, also known as PCSK9, isan enzyme that in humans is encoded by the PCSK9 gene. Seidah et al.,“The secretory proprotein convertase neural apoptosis-regulatedconvertase 1 (NARC-1): liver regeneration and neuronal differentiation”Proc. Natl. Acad. Sci. U.S.A. 100 (3): 928-933 (2003). Similar genes(orthologs) are found across many species. Many enzymes, includingPSCK9, are inactive when they are first synthesized, because they have asection of peptide chains that blocks their activity; proproteinconvertases remove that section to activate the enzyme.

The PSCK9 gene encodes a proprotein convertase belonging to theproteinase K subfamily of the secretory subtilase family. The encodedprotein is synthesized as a soluble zymogen that undergoes autocatalyticintramolecular processing in the endoplasmic reticulum. The protein mayfunction as a proprotein convertase. For example, a human PCSK9 aminoacid sequence is:

001 mgtvssrrsw wplpllllll lllgpagara qededgdyee lvlalrseed glaeapehgt061 tatfhrcakd pwrlpgtyvv vlkeethlsq sertarriqa qaarrgyllk ilhvfhgllp121 gflvkmsgdl lelalklphv dyieedssvf aqsipwnier itppryrade yqppdggslv181 evylldtsiq sdhreiegrv mvtdfenvpe edgtrfhrqa skcdshgthl agvvsgrdag241 vakgasmrsl rvlncqgkgt vsgtliglef irksqlvqpv gplvvllpia ggysrvlnaa301 cqrlaragvv lvtaagnfrd daclyspasa pevitvgatn aqdqpvtlgt lgtnfgrcvd361 lfapgediig assdcstcfv sqsgtsqaaa hvagiaamml saepeltiae lrqrlihfsa421 kdvineawfp edqrvltpnl vaalppsthg agwqlfcrtv wsahsgptrm atavarcapd481 eellscssfs rsgkrrgerm eaqggklvcr ahnafggegv yaiarccllp qancsvhtap541 paeasmgtrv hchqqghvlt gcsshweved lgthkppvlr prgqpnqcvg hreasihasc601 chapgleckv kehgipapqe qvtvaceegw tltgcsalpg tshvigayav dntcvvrsrd661 vsttgstseg avtavaiccr srhlaqasqe lq (Accession No. NP_777596).

PSCK9 is believed to play a regulatory role in cholesterol homeostasis.For example, PCSK9 can bind to the epidermal growth factor-like repeat A(EGF-A) domain of the low-density lipoprotein receptor (LDL-R) resultingin LDL-R internalization and degradation. Clearly, it would be expectedthat reduced LDL-R levels result in decreased metabolism of LDL-C, whichcould lead to hypercholesterolemia.

As it is estimated that approximately 9 million Americans have a high orvery high risk for heart-related problems that could benefit from PCSK9inhibitors (especially when in combination with statins). PCSK9inhibitors could result in such widespread usage having the potential toreplace statins in certain conditions. PCSK9 has medical significancebecause it acts in cholesterol homeostasis. Drugs that block PCSK9biological actions are believed to lower circulating low-densitylipoprotein cholesterol (LDL-C) levels (i.e., for example, by increasingthe availability of LDL-Rs and, consequently, LDL-C clearance). Suchdrugs are beginning Phase III clinical trials to assess their safety andefficacy in humans, and to determine if they can improve outcomes inheart disease.

Drugs that inhibit LDL-R/PCSK9 complex formation have been suggested tolower cholesterol much more than conventionally availablecholesterol-lowering drugs (i.e., for example, statins). It isbiologically plausible that this would also lower heart attacks andother diseases caused by raised cholesterol. Studies with humans,including phase III clinical trials now underway, are focused as towhether PCSK9 inhibition actually does lower cardiovascular disease,with acceptable side effects. Lopez D., “Inhibition of PCSK9 as a novelstrategy for the treatment of hypercholesterolemia” Drug News Perspect.21(6): 323-e30 (2008); Steinberg et al., “Inhibition of PCSK9: apowerful weapon for achieving ideal LDL cholesterol levels” Proc. Natl.Acad. Sci. U.S.A. 106(24): 9546-9547 (2009); Mayer, “Annexin A2 is aC-terminal PCSK9-binding protein that regulates endogenous low densitylipoprotein receptor levels” J. Biol. Chem. 283(46): 31791-31801((2008); and anonymous, “Bristol-Myers Squibb selects Isis drugtargeting PCSK9 as development candidate for prevention and treatment ofcardiovascular disease” Press Release. FierceBiotech. 2008 Apr. 8.

Currently, it has been reported that PCSK9 antibody drugs are inclinical trials (e.g., for example, Sanofi/Regeneron, Amgen, Pfizer,Novartis, Roche). However, one disadvantage of antibody therapy is thatthe administration is performed by subcutaneous or intravenousinjection. A number of monoclonal antibodies that bind to PCSK9 near thecatalytic domain that interact with the LDL-R and hence inhibitLDL-R/PCSK9 complex formation are currently in clinical trials. Theseantibodies include AMG145 (Amgen), 1D05-IgG2 (Merck & Co.), andSAR236553/REGN727 (Aventis/Regeneron). Lambert et al., “The PCSK9decade” J. Lipid Res. 53(12): 2515-2524 (2012).

Peptides that mimic the EGF-A domain of the LDL-R have been developed toinhibit LDL-R/PCSK9 complex formation. Shan et al., “PCSK9 binds tomultiple receptors and can be functionally inhibited by an EGF-Apeptide”. Biochem. Biophys. Res. Commun. 375(1): 69-73 (2008). PeptidicPCSK9 inhibitors of the EGF-A binding site were identified by screeningboth linear and disulfide-constrained phage-displayed peptide libraries.This approach identified a 13-amino acid peptide (Pep2-8) that includesstructural mimicry of the natural binding domain of LDL receptor. Thepeptide inhibitor binding site was determined to largely overlap withthat of the EGF(A) domain; therefore, Pep2-8 acts a competitiveinhibitor of LDL receptor binding. This is akin to the inhibitionmechanism of anti-PCSK9 monoclonal antibodies, which also disrupt theinteraction of the LDL receptor-EGF(A) domain with PCSK9. Zhang et al.,“Identification of a Small Peptide That Inhibits PCSK9 Protein Bindingto the Low Density Lipoprotein Receptor” J Biol Chem 289:942 955 (2014).

PCSK9 antisense oligonucleotides (Isis Pharmaceuticals) have been shownto increase expression of the LDL-R and decrease circulating totalcholesterol levels in mice. Graham et al., “Antisense inhibition ofproprotein convertase subtilisin/kexin type 9 reduces serum LDL inhyperlipidemic mice” J. Lipid Res. 48(4); 763-767 (2007). It has alsobeen reported that a locked nucleic acid (Santaris Pharma) reduced PCSK9mRNA levels in mice. Gupta et al., “A locked nucleic acid antisenseoligonucleotide (LNA) silences PCSK9 and enhances LDLR expression invitro and in vivo” PLoS ONE 5(5): e10682 (2010); and Lindholm et al.,“PCSK9 LNA antisense oligonucleotides induce sustained reduction of LDLcholesterol in nonhuman primates”. Mol. Ther. 20(2):376-381 (2012).Initial clinical trials of an RNAi (ALN-PCS, Alnylam Pharmaceuticals)has shown positive results as an effective means of inhibitingLDL-R/PCSK9 complex formation. Frank-Kamenetsky et al., “TherapeuticRNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents andLDL cholesterol in nonhuman primates” Proc. Natl. Acad. Sci. U.S.A.105(33): 11915-11920 (2008).

II. PCSK9 Allosteric Site Modulation Small Molecule Compounds

Variants of PCSK9 can reduce or increase circulating cholesterol.Abifadel et al., “Mutations in PCSK9 cause autosomal dominanthypercholesterolemia” Nat. Genet. 34 (2): 154-156 (2003). LDL-C isnormally removed from the blood when it binds to an LDL-R on the surfaceof liver cells, and is internalized within the hepatocyte as areceptor-ligand complex. However, when PCSK9 binds to an LDL-R, theLDL-R is concomitantly degraded along with the complexed LDL particle.However, if a PCSK9 is not bound to an LDL-R, the LDL-R is recycledafter internalization thereby returning to the surface of the cell forremoval of more cholesterol.

In some embodiments, the invention relates to small organic compoundshaving a modulation effect on PCSK9's ability to form an LDL-R/PCSK9complex. In some embodiments, the present invention contemplates the useof small organic compounds that bind to a PCSK9 protein and modulate theprotein's biological activity. In some embodiments, the small moleculesdecrease LDL-R/PCSK9 complex formation and are thereby useful to treatvarious diseases comprising lipid dysregulation. In some embodiments,the small molecules increase LDL-R/PCSK9 complex formation and arethereby useful in research and development of therapies relevant to LDLdysregulation.

Although it is not necessary to understand the mechanism of aninvention, it is believed that “gain-of-function” (GOF) PCSK9 mutantsmay result in conditions including, but not limited to,hypercholesterolemia. For example, compounds that bind to a PCSK9 andincrease the affinity of PCSK9's low density lipoprotein receptor for alow density lipoprotein receptor on the surface of a cell (e.g., ahepatocyte) would be expected to increase the symptoms ofhypercholesterolemia by increasing low density lipoprotein receptorinternalization and degradation.

Although it is not necessary to understand the mechanism of aninvention, it is believed that “loss-of-function” (LOF) PCSK9 mutantsmay result in conditions comprising reduced low density lipoproteins andwould be expected to result in hypocholesterolemia thereby reducing therisk of cardiovascular diseases, including but not limited to, coronaryheart disease. For example, small molecule compounds that bind to aPCSK9 that decrease the affinity of PCSK9's low density lipoproteinreceptor binding site for a low density lipoprotein receptor on thesurface of a cell (e.g., a hepatocyte) would be expected to reduce thesymptoms of hypercholesterolemia by promoting low density lipoproteininternalization and clearance due to concomitant recycling of the lowdensity lipoprotein receptor.

The presently disclosed embodiments of PCSK9 binding small organiccompounds have several advantages over current peptides and ligandsdescribed in the art. For example, small molecule PCSK9 bindingcompounds, as contemplated herein, have the advantage that thesecompounds are smaller than many previously described peptides. It isenvisioned that these small organic compounds can be administered orallywithout immunological reactions seen with antibody administration, orsystemic degradation problems as seen with nucleic acid administration(i.e., antisense or locked nucleic acids). Nonetheless, as these smallorganic compounds have long half-lives, encapsulation drug deliverysystems, such as liposomes or other biodegradable protectivecompositions, will lengthen these half-lives to a greater extent thaneither antibodies or nucleic acids. These small organic compoundsdescribed in this application are designed de novo to have desirablecharacteristics, such as for drug-like properties. Although it is notnecessary to understand the mechanism of an invention, it is believedthat these compounds are also structurally distinct from any previouslydescribed PCSK9 modulating small molecules and are reasonably expectedto have different physicochemical properties from other PCSK9 bindingcompounds.

III. Clinical Therapeutics

In some embodiments, the present invention contemplates theadministration of a small molecule PCSK9 allosteric inhibitor compoundto a subject having a symptom of a cardiovascular disease. In oneembodiment, the cardiovascular disease comprises hypercholesterolemia.In one embodiment, the cardiovascular disease comprises hypertension. Inone embodiment, the hypercholesterolemia comprises elevated low densitylipoprotein levels.

In some embodiments, the present invention contemplates theadministration of a small molecule PCSK9 allosteric inhibitor compoundto a subject having a symptom of a metabolic disease. In one embodiment,the metabolic disease comprises diabetes.

Although it is not necessary to understand the mechanism of aninvention, it is believed that the administration of a PCSK9 allostericinhibitor small molecule compound (i.e., such as those described herein)induces a conformational shift of the PCSK9 protein such that theaffinity of the low density lipoprotein binding site for a low densitylipoprotein receptor is decreased, wherein PCSK9/LDL-R complex formationis decreased. The decrease in PCSK9/LDL-R complex formation results inan increase in the bioavailability of LDL-R receptors for binding tocirculating LDL, thereby increasing the internalization and clearance ofLDL by LDL-R. It is further believed that a small molecule PCSK9allosteric inhibitor compound may result in increased bioavailability ofhepatocyte cell LDL-Rs.

Further, although it is not necessary to understand the mechanism of aninvention, it is believed that the administration of a PCSK9 allostericactivator small molecule compound (i.e., such as those described herein)induces a conformational shift of the PCSK9 protein such that theaffinity of the low density lipoprotein binding site for a low densitylipoprotein receptor is increased, wherein PCSK9/LDL-R complex formationis increased or stabilized. The increase or stabilization in PCSK9/LDL-Rcomplex formation results in a decrease in the bioavailability of LDL-Rreceptors for binding to circulating LDL, thereby decreasing theinternalization and clearance of LDL by LDL-R. It is further believedthat a PCSK9 allosteric activator compound may result in decreasedbioavailability of hepatocyte cell LDL-Rs.

A. Hypercholesterolemia

Hypercholesterolemia (also spelled hypercholesterolaemia) is thepresence of high levels of cholesterol in the blood. It is a form of“hyperlipidemia” (elevated levels of lipids in the blood) and“hyperlipoproteinemia” (elevated levels of lipoproteins in the blood).Durrington, P “Dyslipidaemia” The Lancet 362(9385):717-731.Hypercholesterolemia is typically due to a combination of environmentaland genetic factors. Environmental factors include obesity and dietarychoices. Genetic contributions are usually due to the additive effectsof multiple genes, though occasionally may be due to a single genedefect such as in the case of familial hypercholesterolaemia. A numberof secondary causes exist including: diabetes mellitus type 2, obesity,alcohol, monoclonal gammopathy, dialysis, nephrotic syndrome,obstructive jaundice, hypothyroidism, Cushing's syndrome, anorexianervosa, medications (thiazide diuretics, ciclosporin, glucocorticoids,beta blockers, retinoic acid). Bhatnagar et al., (2008)“Hypercholesterolaemia and its management” BMJ 337: a993. Geneticabnormalities are in some cases completely responsible forhypercholesterolemia, such as in familial hypercholesterolemia wherethere is one or more genetic mutations in the autosomal dominant APOBgene, the autosomal recessive LDLRAP1 gene, autosomal dominant familialhypercholesterolemia (HCHOLA3) variant of the PCSK9 gene, or the LDLreceptor gene. “Hypercholesterolemia” Genetics Home Reference U.S.National Institutes of Health,ghr.nlm.nih.gov/condition=hypercholesterolemia. Even when there is nosingle mutation responsible for hypercholesterolemia, geneticpredisposition still plays a major role in combination with sedentarylifestyle, obesity, or an atherogenic diet. Citkowitz et al., (2010)“Polygenic Hypercholesterolemia”. eMedicine Medscape,emedicine.medscape.com/article/121424-overview.

Cholesterol is a sterol. It is one of three major classes of lipidswhich all animal cells utilize to construct their membranes and is thusmanufactured by all animal cells. Plant cells do not manufacturecholesterol. It is also the precursor of the steroid hormones, bileacids and vitamin D. Since cholesterol is insoluble in water, it istransported in the blood plasma within protein particles (lipoproteins).Lipoproteins are classified by their density: very low densitylipoprotein (VLDL), intermediate density lipoprotein (IDL), low densitylipoprotein (LDL) and high density lipoprotein (HDL). Biggerstaff etal., (2004). “Understanding lipoproteins as transporters of cholesteroland other lipids” Adv Physiol Educ 28 (1-4): 105-6. All the lipoproteinscarry cholesterol, but elevated levels of the lipoproteins other thanHDL (termed non-HDL cholesterol), particularly LDL-cholesterol areassociated with an increased risk of atherosclerosis and coronary heartdisease. Carmena et al., (2004) “Atherogenic lipoprotein particles inatherosclerosis” Circulation 109 (23 Suppl 1): III 2-7. In contrast,higher levels of HDL cholesterol are protective. Kontush et al., (2006)“Antiatherogenic small, dense HDL-guardian angel of the arterial wall?”Nat Clin Pract Cardiovasc Med 3(3):144-153. Elevated levels of non-HDLcholesterol and LDL in the blood may be a consequence of diet, obesity,inherited (genetic) diseases (such as LDL receptor mutations in familialhypercholesterolemia), or the presence of other diseases such asdiabetes and an underactive thyroid. Total cholesterol is the amount ofall of the fats in your blood. These fats are called lipids. There aredifferent types of lipid that make up your total cholesterol. The twomost important types are: low density lipoprotein (LDL)—“bad”cholesterol and high density lipoprotein (HDL)—“good” cholesterol. Highcholesterol, especially “bad” cholesterol (LDL), can clog your arteries.This may reduce blood flow to your heart. It can lead to heart disease,stroke, or heart attack. Cholesterol is measured in milligrams perdeciliter (mg/dL). In conditions such as heart disease or diabetes, LDLcholesterol should stay below 100 mg/dL. If there is a risk for heartdisease, LDL cholesterol should be lower than 130 mg/dL. In general, LDLcholesterol should be lower than 160-190 mg/dL. Alternative, HDL “good”cholesterol should be high. For example, HDL levels in men should beabove 40 mg/dL, while HDL levels should be above 50 mg/dL for women.

One symptom of hypercholesterolemia comprises a longstanding elevationof serum cholesterol that can lead to atherosclerosis. Bhatnagar et al.,(2008) “Hypercholesterolaemia and its management” BMJ 337: a993. Over aperiod of decades, chronically elevated serum cholesterol contributes toformation of atheromatous plaques in the arteries. This can lead toprogressive stenosis (narrowing) or even complete occlusion (blockage)of the involved arteries. Alternatively smaller plaques may rupture andcause a clot to form and obstruct blood flow. Finn A V, Nakano M, NarulaJ, Kolodgie F D, Virmani R (July 2010). “Concept of vulnerable/unstableplaque” Arterioscler. Thromb. Vasc. Biol. 30(7): 1282-1292. A suddenocclusion of a coronary artery results in a myocardial infarction orheart attack. An occlusion of an artery supplying the brain can cause astroke. If the development of the stenosis or occlusion is gradual bloodsupply to the tissues and organs slowly diminishes until organ functionbecomes impaired. At this point that tissue ischemia (restriction inblood supply) may manifest as specific symptoms including, but notlimited to, temporary ischemia of the brain (commonly referred to as atransient ischemic attack) may manifest as temporary loss of vision,dizziness and impairment of balance, aphasia (difficulty speaking),paresis (weakness) and paresthesia (numbness or tingling), usually onone side of the body. Insufficient blood supply to the heart maymanifest as chest pain, and ischemia of the eye may manifest astransient visual loss in one eye. Insufficient blood supply to the legsmay manifest as calf pain when walking, while in the intestines it maypresent as abdominal pain after eating a meal. Grundy et al., (1998)“Primary prevention of coronary heart disease: guidance from Framingham:a statement for healthcare professionals from the AHA Task Force on RiskReduction. American Heart Association” Circulation 97(18):1876-1887.

B. Hypocholesterolemia

Hypocholesterolemia is the presence of abnormally low (hypo-) levels ofcholesterol in the blood (-emia). Although the presence of high totalcholesterol (hyper-cholesterolemia) correlates with cardiovasculardisease, a defect in the body's production of cholesterol can lead toadverse consequences as well. Cholesterol is an essential component ofmammalian cell membranes and is required to establish proper membranepermeability and fluidity. It is not clear if a lower than averagecholesterol level is directly harmful; it is often encountered inparticular illnesses.

Possible causes of low cholesterol include, but are not limited to,statins, hyperthyroidism, or an overactive thyroid gland, adrenalinsufficiency, liver disease, malabsorption (inadequate absorption ofnutrients from the intestines), such as in celiac disease, malnutrition,abetalipoproteinemia (a genetic disease that causes cholesterol readingsbelow 50 mg/dl), hypobetalipoproteinemia (a genetic disease that causescholesterol readings below 50 mg/dl, manganese deficiency,Smith-Lemli-Opitz syndrome, Marfan syndrome, leukemias and otherhematological diseases.

Demographic studies suggest that low cholesterol is associated withincreased mortality, mainly due to depression, cancer, hemorrhagicstroke, aortic dissection and respiratory diseases. Jacobs et al.,(1992). “Report of the Conference on Low Blood Cholesterol: MortalityAssociations” Circulation 86 (3): 1046-1060; and Suarez E. C., (1999)“Relations of trait depression and anxiety to low lipid and lipoproteinconcentrations in healthy young adult women”. Psychoson Med 61(3):273-279. It is also possible that whatever causes the low cholesterollevel also causes mortality, and that the low cholesterol is simply amarker of poor health.

C. Diabetes

Diabetes affects more than 20 million Americans. Over 40 millionAmericans have pre-diabetes (which often develops before type 2diabetes). Diabetes is usually a lifelong (chronic) disease in whichthere is a high level of sugar in the blood. Insulin is a hormoneproduced by the pancreas to control blood sugar. Diabetes can be causedby too little insulin, resistance to insulin, or both. To understanddiabetes, it is important to first understand the normal process bywhich food is broken down and used by the body for energy.

Several things happen when food is digested. A sugar called glucoseenters the bloodstream. Glucose is a source of fuel for the body. Anorgan called the pancreas makes insulin. The role of insulin is to moveglucose from the bloodstream into muscle, fat, and liver cells, where itcan be used as fuel.

People with diabetes have high blood sugar because their body cannotmove sugar into fat, liver, and muscle cells to be stored for energy.This is because either their pancreas does not make enough insulin ortheir cells do not respond to insulin normally.

There are two major types of diabetes. The causes and risk factors aredifferent for each type. Type 1 diabetes can occur at any age, but it ismost often diagnosed in children, teens, or young adults. In thisdisease, the body makes little or no insulin. Daily injections ofinsulin are needed. The exact cause is unknown. Type 2 diabetes makes upmost diabetes cases. It most often occurs in adulthood. But because ofhigh obesity rates, teens and young adults are now being diagnosed withit. Many people with type 2 diabetes do not know they have it.

Gestational diabetes is high blood sugar that develops at any timeduring pregnancy in a woman who does not have diabetes.

Diabetes symptoms may result from high blood sugar level and include,but are not limited to, blurry vision, excess thirst, fatigue, hunger,urinating often and weight loss.

IV. Pharmaceutical Compositions

The present invention further provides pharmaceutical compositions(e.g., comprising the compounds described above). The pharmaceuticalcompositions of the present invention may be administered in a number ofways depending upon whether local or systemic treatment is desired andupon the area to be treated. Administration may be topical (includingophthalmic and to mucous membranes including vaginal and rectaldelivery), pulmonary (e.g., by inhalation or insufflation of powders oraerosols, including by nebulizer; intratracheal, intranasal, epidermaland transdermal), oral or parenteral. Parenteral administration includesintravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial, (e.g., intrathecalor intraventricular), administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

Compositions and formulations for oral, sublingual or buccaladministration include powders or granules, suspensions or solutions inwater or non-aqueous media, capsules, gels, drops, strips, gums, sachetsor tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

In some embodiment, the pharmaceutical compositions may further compriseother drugs and/or hormones. For example, the pharmaceutical compositionmay further comprise a statin drug. Statins (or HMG-CoA reductaseinhibitors) are a class of drugs used to lower cholesterol levels byinhibiting the enzyme HMG-CoA reductase, which plays a role in theproduction of cholesterol in the liver. Increased cholesterol levelshave been associated with cardiovascular diseases, and statins aretherefore used in the prevention of these diseases. Lewington et al.,“Blood cholesterol and vascular mortality by age, sex, and bloodpressure: a meta-analysis of individual data from 61 prospective studieswith 55,000 vascular deaths” Lancet 370(9602): 1829-1839 (2007).Research has found that statins are most effective for treatingcardiovascular disease (CVD) as a secondary prevention strategy, withquestionable benefit in those with elevated cholesterol levels butwithout previous CVD. Taylor et al. “Statins for the primary preventionof cardiovascular disease”. In; Taylor, Fiona. Cochrane Database SystRev (1) (2011). Statins have rare but severe adverse effects,particularly muscle damage.

Specific examples of statins include, but are not limited to,atorvastatin (Lipitor® and Torvast®), fluvastatin (Lescol®), lovastatin(Mevacor®, Altocor®, Altoprev®), pitavastatin (Livalo®, Pitava®),pravastatin (Pravachol®, Selektine®, Lipostat®), rosuvastatin (Crestor®)and simvastatin (Zocor®, Lipex®). Several combination preparations of astatin and another agent, such as ezetimibe/simvastatin, are alsoavailable.

Specific examples of cardiovascular drugs include, but are not limitedto, propranolol, digitalis, amlodipine besylate, and nifedipine.

Specific examples of other pharmaceutical compositions may furtherinclude, but are not limited to, exetimibe (Zetia®), amlodipine besylate(Norvasc®), niacin, sitagliptin (Januvia®), metformin or orlistat(Alli®/Xenical®).

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances that increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the active pharmaceutical ingredient(s) of theformulation.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient. Theadministering physician can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC_(50S) found to be effective in invitro and in vivo animal models or based on the small molecule compoundsdescribed herein. In general, dosage is from 0.01 μg to 100 g per kg ofbody weight, and may be given once or more daily, weekly, monthly oryearly. The treating physician can estimate repetition rates for dosingbased on measured residence times and concentrations of the drug inbodily fluids or tissues. Following successful treatment, it may bedesirable to have the subject undergo maintenance therapy to prevent therecurrence of the disease state, wherein the small molecule compound isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

In one embodiment, the present invention further contemplates acommercial package comprising (a) a pharmaceutical compositioncomprising a small molecule compound as contemplated herein; and (b)instructions for the use thereof for treatment of hypercholesterolemia.In one embodiment, the present invention further contemplates acommercial package comprising (a) a pharmaceutical compositioncomprising a small molecule compound as contemplated herein; and (b)instructions for the use thereof for treatment of hypocholesterolemia.In one embodiment, the present invention further contemplates acommercial package comprising (a) a pharmaceutical compositioncomprising a small molecule compound as contemplated herein; and (b)instructions for the use thereof for inhibition of PCSK9 proteinbiological activity. In one embodiment, the present invention furthercontemplates a commercial package comprising (a) a pharmaceuticalcomposition comprising a small molecule compound as contemplated herein;and (b) instructions for the use thereof for increasing the biologicalactivity of PCSK9 protein. In one embodiment, the present inventionfurther contemplates a commercial package as a kit.

In one embodiment, the present invention further contemplates a kitcomprising (a) a pharmaceutical composition comprising a small moleculecompound as contemplated herein; and (b) instructions for the usethereof for treatment of hypercholesterolemia. In one embodiment, thepresent invention further contemplates a kit comprising (a) apharmaceutical composition comprising a small molecule compound ascontemplated herein; and (b) instructions for the use thereof fortreatment of hypocholesterolemia. In one embodiment, the presentinvention further contemplates a kit comprising (a) a pharmaceuticalcomposition comprising a small molecule compound as contemplated herein;and (b) instructions for the use thereof for inhibition of PCSK9 proteinbiological activity. In one embodiment, the present invention furthercontemplates a kit comprising (a) a pharmaceutical compositioncomprising a small molecule compound as contemplated herein; and (b)instructions for the use thereof for increasing the biological activityof PCSK9 protein.

V. Description of Chemistry Phenylpiperazine Scaffold—Synthesis:

The sulfonamido compounds of a phenylpiperazine scaffold of Formula Icould be prepared using standard synthetic methods (Scheme 1):

Related analogs are accessible by a variety of known procedures (Scheme2).

Other, heterocyclic analogs can be made according to Scheme 3:

Piperidine analogs may be obtained by chemistry such as indicated inScheme 4:

EXAMPLES

The following examples are included to demonstrate specific embodimentsof the disclosure. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques to function well in the practice of the disclosure, and thuscan be considered to constitute specific modes for its practice.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the disclosure.

Compound Synthesis

Step 1: Synthesis of 4-bromo-N-phenylbenzenesulfonamide

4-Bromobenzenesulfonyl chloride (2.00 g, 7.82 mmol, 1.0 eq), aniline(0.87 g, 9.34 mmol, 1.2 eq) and pyridine (3.00 g, 39.06 mmol, 5.0 eq)were dissolved in dichloromethane (25 mL) under nitrogen atmosphere andthe solution was stirred at ambient temperature for 6 h. After completeconsumption of starting material, the reaction mixture was diluted withdichloromethane and washed with 0.5 N aq HCl followed by water,saturated aq NaHCO₃ and brine. The organic extract was then dried overanhydrous sodium sulfate, filtered, and solvent evaporated from thefiltrated under reduced pressure to afford4-bromo-N-phenylbenzenesulfonamide as yellow solid (1.4 g, 57.3%). LCMS:Purity 99.69%. MS calculated for [M] 310.96 and found [M−H]⁺ 309.88.

Step 2: Synthesis of tert-butyl4-(4-(N-phenylsulfamoyl)phenyl)piperazine-1-carboxylate

A mixture of 4-bromo-N-phenylbenzenesulfonamide (1.00 g, 3.20 mmol, 1.0eq), tert-butyl piperazine-1-carboxylate (0.71 g, 3.81 mmol, 1.2 eq),K₂CO₃ (0.89 g, 6.40 mmol, 2.0 eq), CuI (0.12 g, 0.640 mmol, 0.2 eq) andL-proline (0.11 g, 0.955 mmol, 0.3 eq) in DMSO (15 mL) was stirred at90° C. for 18 h. After complete consumption of starting material, themixture was cooled to ambient temperature and partitioned between waterand ethyl acetate. The organic extract was separated and the aqueousextract was again extracted with ethyl acetate. The combined organicextract was washed with brine, dried over anhydrous Na₂SO₄, filtered andsolvents evaporated from the filtrate under reduced pressure to obtain acrude, which was purified by flash chromatography on silica gel, 230-400mesh, using gradient of ethyl acetate in hexanes as eluent to obtaintert-butyl 4-(4-(N-phenylsulfamoyl)phenyl)piperazine-1-carboxylate asyellow solid, yield (0.25 g, 18.7%). LCMS: Purity 84.26%. MS calculatedfor [M] 417.17 and found [M−H]⁺ 416.08.

Step 3: Synthesis of N-phenyl-4-(piperazin-1-yl)benzenesulfonamide

Trifluoroacetic acid (0.314 g, 2.75 mmol, 5.0 eq) was added to asolution of tert-butyl4-(4-(N-phenylsulfamoyl)phenyl)piperazine-1-carboxylate (0.23 g, 0.55mmol, 1.0 eq) in dichloromethane (10 mL) at ambient temperature and themixture was stirred for 3 h. After complete consumption of startingmaterial, solvents evaporated from the mixture under reduced pressure,the residue was diluted with aq. sodium bicarbonate and extracted with amixture of methanol and dichloromethane (1:20). The combined organicextract was washed with brine, dried over anhydrous Na₂SO₄, filtered andsolvents evaporated from the filtrate under reduced pressure to obtainN-phenyl-4-(piperazin-1-yl)benzenesulfonamide as off white solid (0.078g, 93%). LCMS: Purity 98.69%. MS calculated for [M] 317.12 and found[M+H]⁺ 318.05.

Step 4: Synthesis of N-phenyl-4-(piperazin-1-yl)benzenesulfonamidehydrochloride

4M HCl in 1,4-dioxane (2.0 mL) was added to a solution ofN-phenyl-4-(piperazin-1-yl) benzene sulfonamide (0.03 g, 0.094 mmol, 1.0eq) in dichloromethane (10 mL) at ambient temperature and the mixturewas stirred 2 h. The solvent was evaporated under reduced pressure, theresidue was triturated with diethyl ether, filtered and dried undervacuum to afford N-phenyl-4-(piperazin-1-yl) benzene sulfonamidehydrochloride as off white solid (0.022 g, 61%). LCMS: Purity 98.78%. MScalculated for [M] 317.12 and found [M+H]⁺ 318.41. ¹H NMR (400 MHz,DMSO-d₆) δ: 10.07 (s, 1H), 9.07 (s, 2H), 7.58 (d, J 9.0 Hz, 2H), 7.21(t, J=8.2 Hz, 2H), 7.08 (d, J=7.6 Hz, 2H), 7.03 (d, J=9.1 Hz, 2H), 6.99(t, J=7.4 Hz, 1H), 3.50 (t, J=4.8 Hz, 4H), 3.17 (bs, 4H).

Step 1: Synthesis of4-bromo-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide

4-Bromobenzenesulfonyl chloride (3.00 g, 11.70 mmol, 1.0 eq),5,6,7,8-tetrahydronaphthalen-2-amine (2.07 g, 14.08 mmol, 1.2 eq) andpyridine (3.00 g, 39.06 mmol, 5.0 eq) were dissolved in dichloromethane(30 mL) under nitrogen atmosphere and the solution was stirred atambient temperature for 3 h. After complete consumption of startingmaterial, the reaction mixture was diluted with dichloromethane andwashed with 0.5 N aq HCl followed by water, saturated aq NaHCO₃ andbrine. The organic extract was then dried over anhydrous sodium sulfate,filtered, and solvent evaporated from the filtrated under reducedpressure to 4-bromo-N-(5,6,7,8-tetrahydronaphthalen-2-yl) benzenesulfonamide as yellow sticky mass (2.2 g, 51.1%). LCMS: Purity 81.44%.MS calculated for [M] 365.01 and found [M−H]⁺ 363.92.

Step 2: Synthesis of tert-butyl4-(4-(N-(5,6,7,8-tetrahydronaphthalen-2-yl)sulfamoyl)phenyl)piperazine-1-carboxylate

A mixture of 4-bromo-N-(5,6,7,8-tetrahydronaphthalen-2-yl) benzenesulfonamide (2.00 g, 5.47 mmol, 1.0 eq), tert-butylpiperazine-1-carboxylate (1.2 g, 6.57 mmol, 1.2 eq), K₂CO₃ (3.48 g,16.39 mmol, 3.0 eq), Pd₂(dba)₃ (0.23 g, 0.22 mmol, 0.04 eq) andrec-BINAP (0.48 g, 0.77 mmol, 0.14 eq) in 1,4-Dioxane (25.0 mL) wasstirred at 100° C. for 18 h. After complete consumption of startingmaterial, the mixture was cooled to ambient temperature and partitionedbetween water and ethyl acetate. The organic extract was separated andthe aqueous extract was again extracted with ethyl acetate. The combinedorganic extract was washed with brine, dried over anhydrous Na₂SO₄,filtered and solvents evaporated from the filtrate under reducedpressure to obtain a crude, which was purified by flash chromatographyon silica gel, 230-400 mesh, using gradient of ethyl acetate in hexanesas eluent to obtain tert-butyl4-(4-(N-(5,6,7,8-tetrahydronaphthalen-2-yl)sulfamoyl)phenyl)piperazine-1-carboxylateas yellow solid (0.35 g, 14%). LCMS: Purity 80.35%. MS calculated for[M] 471.22 and found [M+H]⁺ 472.29.

Step 3: Synthesis of4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide

Trifluoroacetic acid (0.36 g, 3.18 mmol, 5.0 eq) was added to a solutionof tert-butyl4-(4-(N-(5,6,7,8-tetrahydronaphthalen-2-yl)sulfamoyl)phenyl)piperazine-1-carboxylate(0.30 g, 0.64 mmol, 1.0 eq) in dichloromethane (10 mL) at ambienttemperature and the mixture was stirred for 2 h. After completeconsumption of starting material, solvents evaporated from the mixtureunder reduced pressure, the residue was diluted with aq. sodiumbicarbonate and extracted with dichloromethane. The combined organicextract was washed with brine, dried over anhydrous Na₂SO₄, filtered andsolvents evaporated from the filtrate under reduced pressure to obtain4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamideas off white solid (0.052 g, 21.1%). LCMS: Purity 98.23%. MS calculatedfor [M] 371.17 and found [M+H]⁺ 371.10.

Step 4: Synthesis of4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamidehydrochloride

4M HCl in 1,4-dioxane (2.5 mL) was added to a solution of4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide(0.05 g, 0.13 mmol, 1.0 eq) in dichloromethane (10 mL) at ambienttemperature and the mixture was stirred 2 h. The solvent was evaporatedunder reduced pressure, the residue was triturated with diethyl ether,filtered and dried under vacuum to afford4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamidehydrochloride as off white solid (0.022 g, 61%). LCMS. Purity 99.59/o.MS calculated for [M]371.17 and found [M+H]⁺ 372.49. ¹H NMR (400 MHz,DMSO-d₆) δ: 9.85 (s, 1H), 9.06 (s, 2H), 7.58 (d, J=9.0 Hz, 2H), 7.04 (d,J=9.0 Hz, 2H), 6.86 (d, J=8.2 Hz, 1H), 6.80 (d, J=6.1 Hz & 2.1 Hz, 1H),6.76 (d, J=1.8 Hz, 1H), 3.50 (t, J=4.8 Hz, 4H), 3.17 (t, J=4.8 Hz, 4H),2.57 (bs, 4H), 1.65 (bs, 4H).

Preparation of N-phenyl-4-(piperidin-4-yl)benzenesulfonamide

The title compound was prepared by a method similar to Scheme 4. MScalculated for [M]316.12 and found [M+H]⁺ 317.16.

Preparation of4-(piperidin-4-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide

The title compound was prepared by a method similar to Scheme 4. MScalculated for [M]370.17 and found [M+H]⁺ 371.23.

Biological Assays

The compounds of the present disclosure may be tested for binding to,inhibition of, and/or modulation of PCSK9 activity according to thefollowing protocols.

Cell Culture

Cells, such as HepG2, HuH7, FL83B, or a cell line transfected with ashort-hairpin PCSK9 knockdown sequence (e.g., HepG2/shPCSK9,HuH7/shPCSK9) can be cultured following routine procedures, such asthose described by Benjannet et al., “Effects of the prosegment and pHon the activity of PCSK9: evidence for additional processing events”JBiol Chem. 285(52): 40965-40978 (2010), which is hereby incorporated byreference in its entirety.

LDLR Flow Cytometric Analysis

LDLR levels can be measured using flow cytometry or fluorescenceactivated cell sorting (FACS) using protocols adapted from Benjannet etal., “Effects of the prosegment and pH on the activity of PCSK9:evidence for additional processing events” J Biol Chem. 285(52):40965-40978 (2010) and “Composition and Methods of Use of SmallMolecules as Binding Ligands for the Modulation of Proprotein ConvertaseSubtilisin/Kexin Type 9 (PCSK9) Protein Activity” (WO2016029037), whichare incorporated by reference in their entirety.

Cells, such as HepG2, HuH7, FL83B, or a cell line transfected with ashort-hairpin PCSK9 knockdown sequence such as HepG2/shPCSK9,HuH7/shPCSK9, or FL83B/shPCSK9 are plated and cultured at 37° C. for12-24 h. Culture media is removed and replaced with fresh culture mediaor culture media plus a predetermined amount of recombinant PCSK9, forexample a 10 nM final concentration of PCSK9. Cell culture media can becomposed of DMEM (Invitrogen) with 10% fetal bovine serum (LifeTechnologies) supplemented with penicillin-streptomycin (LifeTechnologies). Cells are dosed with small molecule test compounds atdoses ranging from 0 nM to 100 uM

Following an incubation period of specified length, such as 6 hours, themedia is removed and the cells are rinsed three times with a rinsesolution (i.e., Dulbecco's phosphate buffered saline (D-PBS, LifeTechnologies) supplemented with 0.5% bovine serum albumin (BSA, Sigma)and 1 g/L glucose (Sigma)). The fluid is then removed, and cells arereleased from the plate using TrypLE Express (Life Technologies) permanufacturer's recommend procedures, such as incubation for 5-10 minutesat 37° C. The TyrpLE-Cell suspension is then transferred to 15 mLconical tubes, volume is increased to 2 mL with D-PBS supplemented with0.5% BSA and 1 g/mL glucose, and the tubes are centrifuged at 250×gravity for 10 minutes. Following centrifugation, the supernatant isaspirated and the cell pellet is resuspended in 100 uL D-PBS containing0.5% BSA and 1 g/mL glucose, and cells are labeled with anti-LDLRantibody per the manufacturer's recommended procedure. The cells arethen pelleted by centrifugation, resuspended in 300 uL PBS andcounterstained with 4′,6-Diamidino-2-phenylindole (DAPI, CaymanChemical) as a cell viability marker, other cell viability markers suchas 7-aminoactinomycin D (7AAD, Life Technologies) have also beendescribed in the art.

Cells are analyzed for both cell viability marker (dead cells) and LDLRin live cells using a flow cytometer per the manufacturer's operatingmanual. Cells incubated with small molecule compounds that areinhibitors of PCSK9 will be expected to show increased amounts of LDLR,relative to control (no compound) specimens, and cells incubated withsmall molecule compounds that are activators of PCSK9 will be expectedto show decreased amounts of LDLR relative to control (no compound)specimens.

Cellular DiI-LDL Uptake Analysis

Cellular DiI-LDL uptake can be measured using protocols adapted fromBenjannet et al., “Effects of the prosegment and pH on the activity ofPCSK9: evidence for additional processing events” J Biol Chem. 285(52):40965-40978 (2010) and “Composition and Methods of Use of SmallMolecules as Binding Ligands for the Modulation of Proprotein ConvertaseSubtilisin/Kexin Type 9 (PCSK9) Protein Activity” (WO2016029037), whichare incorporated by reference in their entirety.

Cells, such as HepG2, HuH7, FL83B, or a cell line transfected with ashort-hairpin PCSK9 knockdown sequence such as HepG2/shPCSK9,HuH7/shPCSK9, or FL83B/shPCSK9 are plated and cultured at 37° C. for12-24 h. Culture media is removed and replaced with freshlipoprotein-depleted culture media supplemented with 5 ug/mL of DiI-LDL(Kalen Biomedical) or lipoprotein-depleted culture media supplementedwith 5 ug/mL of DiI-LDL plus a predetermined concentration ofrecombinant PCSK9, for example a 10 nM final concentration of PCSK9.Lipoprotein-depleted culture media can be composed of DMEM (Invitrogen)with 10% lipoprotein-depleted fetal bovine serum (Kalen Biomedical) andsupplemented with penicillin-streptomycin (Life Technologies). Cells aredosed with small molecule test compounds at doses ranging from 0 nM to100 uM.

Following an incubation period of specified length, such as 6 hours,Hoechst 33342 (AnaSpec) stain is added to the cell media permanufacturer's instructions and incubated for a specified length (e.g.,30 minutes). The lipoprotein-depleted media is removed and cells rinsedthree times with phosphate buffered saline. A final volume of phosphatebuffered saline is added back to the wells. The DiI fluorescence ismeasured with a plate reader using an exciting wavelength of 550 nm andthe resulting emission at 590 nm is measured. The Hoechst stainfluorescence is measured with a plate reader using an excitingwavelength of 355 nm and the resulting emission at 460 nm is measured.

Cells are analyzed by for both Hoechst stain (DNA content) and DiI-LDLfluorescence. Cells incubated with small molecule compounds that areinhibitors of PCSK9 will be expected to show increased amounts ofDiI-LDL fluorescence, relative to control (no compound) specimens, andcells incubated with small molecule compounds that are activators ofPCSK9 will be expected to show decreased amounts of DiI-LDL fluorescencerelative to control (no compound) specimens.

LDL Uptake Cell-Based Assay Kit

LDL uptake and LDLR expression can also be measured in cells, such asHepG2 or HuH7 cells, using a commercial kit (Cayman Chemical Co.,Catalog #10011125) and the accompanying protocols provided by themanufacturer.

Back-Scattering Interferometry Direct Binding Measurement

Direct binding can be measured using Back-Scattering Interferometry(BSI), which has been previously described in “Interferometric detectionsystem and method” (EP 1210581), “Free solution measurement of molecularinteractions by backscattering interferometry” (WO 2009039466),“Temperature-stable interferometer” (WO 2009076372), and “Improved eventdetection for back-scattering interferometry” (WO 2013158300); each ofwhich are hereby incorporated by reference in their entirety.

Representative Table of Cell Activity of PCSK9 Modulating Compounds

TABLE 1 Representative Cell Activity of PCSK9 Inhibitory Compounds LDLRFlow Uptake Assay: Assay: % Inhibition % Inhibition of 2.5 ug/mL of 5.0ug/mL recombinant recombinant Com- PCSK9 @ X PCSK9 @ X pound # StructureIUPAC Chemical Name compound dose compound dose Com- pound 1

1-(4-(benzyloxy)phenyl) piperazine >100% @ 10 uM ~16% @ 1.25 uM, ~34% @10 uM Com- pound 2

1-(2-fluoro-4-(phenyl- sulfonyl)phenyl) piperazine >100% @ 20 uM ~45% @1.25 uM, ~66% @ 5 uM Com- pound 3

1-(4-(phenoxyphenyl) piperazine >100% @ 10 uM ~20% @ 1.25 uM, ~30% @ 10uM Com- pound 4

6-(4-(piperazin-1-yl) phenoxy)pyrimidin- 4-ol ~19% @ 0.313 uM, ~30% @1.25 uM Com- pound 5

1-(4-(pyrrolidin-1- ylsulfonyl)phenyl) piperazine ~10% @ 1.25 uM, ~20% @10 uM Com- pound 6

1-(4-(4-fluorobenzyl) phenyl)piperazine >100% @ 5 uM ~15% @ 1.25 uM,~40% @ 10 uM Com- pound 7

1-(4-phenoxyphenyl) piperazin-2-one ~18% @ 1.25 uM Com- pound 8

4-(4-(cyclohexyloxy) phenyl)piperidine >100% @ 5 uM ~15% @ 0.313 uM,~25% @ 1.25 uM ~35% @ 10 uM Com- pound 9

4-(4-((tetrahydro-2H- pyran-4-yl)oxy) phenyl)piperidine ~23% @ 20 uMCom- pound 10

N-phenyl-4- (piperazin-1-yl) benzenesulfonamide >100% @ 20 uM >10% @1.25 uM, >25% @ 10 uM, >40% @ 20 uM Com- pound 11

N-phenyl-4- (piperazin-1-yl) benzamide ~49% @ 20 uM Com- pound 12

4-(piperazin-1-yl)- N-(5,6,7,8-tetra- hydronaphthalen- 2-yl)benzenesulfonamide ~66% @ 1.25 uM, >100% @ 2.5 uM ~10% @ 1.25 uM, ~25% @10 uM, ~45% @ 20 uM Com- pound 13

1-(4-(cyclohexyloxy) phenyl)piperazine >100% @ 20 uM ~20% @ 0.625 uM,~40% @ 10 uM Com- pound 14

N-phenyl-4- (piperidin-4-yl) benzenesulfonamide >100% @ 20 uM Com- pound15

4-(piperidin-4-yl)- N-(5,6,7,8-tetra- hydronaphthalen- 2-yl)benzenesulfonamide >50% @ 1.25 uM, >100% @ 2.5 uM

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains.

The disclosed embodiments illustratively described herein may suitablybe practiced in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising”, “including,” “containing”, etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the claims.

Thus, it should be understood that although the present disclosure hasbeen specifically disclosed by exemplary embodiments and optionalfeatures, modification, improvement and variation of the disclosedembodiments may be implemented by those skilled in the art, and thatsuch modifications, improvements and variations are considered to bewithin the scope of the present disclosure and claims. The materials,methods, and examples provided here are representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the disclosure nor as limitations on the scope of the appendedclaims.

The disclosure has been described broadly and generically herein. Eachof the narrower species and subgeneric groupings falling within thegeneric disclosure also form part of the disclosure. This includes thegeneric description of the disclosure with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

It is to be understood that while the disclosure has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of thedisclosure. Other aspects, advantages and modifications within the scopeof the disclosure will be apparent to those skilled in the art to whichthe disclosure pertains.

1.-12. (canceled)
 13. A method of inhibiting PCSK9 in a subject with ametabolic disease, wherein the method comprises administering to thesubject a compound of formula I:

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein: A is —SO₂— and B is —NH—; or A is —O— or —CH₂— and B is —CH₂—or absent; ring C is a 5 or 6 membered heteroaryl or heterocyclic ring,or a 6 membered aryl ring; E¹, E², and E³ are independently selectedfrom C, CH, and N, wherein one of E¹, E², and E³ may be absent; R¹ andR² are independently selected from the group consisting of H, loweralkyl, hydroxy, amino, aminoalkyl, hydroxyalkyl, haloalkyl, carboxy,—CONH₂, —CON-alkyl, nitrile, —S-alkyl, —O-alkyl, acyl, and oxo; or R¹and R² with the atoms attached thereto form a 5-6 membered fused aryl,heteroaryl, carbocyclic or heterocyclic ring D containing 0-3heteroatoms, where ring D may further be substituted at a position twoatoms away from the juncture with ring C; R³ is independently selectedfrom the group consisting of H, lower alkyl, hydroxy, amino, aminoalkyl,hydroxyalkyl, haloalkyl, carboxy, —CONH₂, —CON-alkyl, nitrile, —S-alkyl,—O-alkyl, acyl, and oxo; R⁴ is selected from the group consisting of H,OH, halogen, and lower alkyl; each R⁵ is hydrogen or taken together areoxo; Y is selected from the group consisting of N and CH; and X¹, X²,X³, and X⁴ are independently selected from the group consisting of CH,C—R⁴, and N.
 14. The method of claim 13, wherein an uptake of LDL byhepatocytes in the subject is modulated.
 15. The method of claim 13,wherein said metabolic disease is diabetes.
 16. The method of claim 13,wherein the compound of formula I is formulated as a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier orexcipient.
 17. The method of claim 16, wherein said pharmaceuticalcomposition further comprises a second pharmaceutical drug compound. 18.The method of claim 17, wherein said second pharmaceutical drug compoundis selected from the group consisting of a statin, a cardiovasculardrug, a metabolic drug, and an antihypertensive drug.
 19. The method ofclaim 13, wherein the compound is selected from the group consisting of:N-phenyl-4-(piperazin-1-yl)benzenesulfonamide;N-isopropyl-4-((4-(piperazin-1-yl)phenyl)sulfonamido)benzamide;N-methyl-4-((4-(piperazin-1-yl)phenyl)sulfonamido)benzamide;N-(3,4-dimethylphenyl)-4-(piperazin-1-yl)benzenesulfonamide;N-(3,5-dimethylphenyl)-4-(piperazin-1-yl)benzenesulfonamide;N-(3,5-dichlorophenyl)-4-(piperazin-1-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;N-(naphthalen-2-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(cinnolin-7-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(isoquinolin-7-yl)-4-(piperazin-1-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(quinolin-6-yl)benzenesulfonamide;N-(cinnolin-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(quinoxalin-6-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(pyrido[2,3-b]pyrazin-7-yl)benzenesulfonamide;N-(1H-benzo[d][1,2,3]triazol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(2-methylbenzo[b]thiophen-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(benzo[c][1,2,5]thiadiazol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(1H-indol-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(1H-indol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(benzo[d]isoxazol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(2-aminoimidazo[1,2-a]pyrimidin-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(2,3-dihydrobenzofuran-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(2,3-dihydrobenzofuran-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;2-hydroxy-N-phenyl-4-(piperazin-1-yl)benzenesulfonamide;4-(2-oxopiperazin-1-yl)-N-phenylbenzenesulfonamide;2-fluoro-4-(2-oxopiperazin-1-yl)-N-phenylbenzenesulfonamide;N-phenyl-4-(piperazin-1-yl-2,2,6,6-d₄)benzenesulfonamide;N-phenyl-4-(piperidin-4-yl)benzenesulfonamide;4-(piperidin-4-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(pyridin-4-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(pyridin-3-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(pyridin-2-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(thiazol-2-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(quinolin-7-yl)benzenesulfonamide;N-phenyl-6-(piperazin-1-yl)pyridine-3-sulfonamide;N-phenyl-5-(piperazin-1-yl)pyridine-2-sulfonamide;N-(3,5-difluorophenyl)-4-(piperazin-1-yl)benzenesulfonamide;3-fluoro-4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;6-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)pyridine-3-sulfonamide;5-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)pyridine-2-sulfonamide;N-phenyl-4-(piperidin-4-yl)benzenesulfonamide; and4-(piperidin-4-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;or a pharmaceutically acceptable salt thereof.
 20. The method of claim13, wherein the compound is selected from the group consisting of:1-(4-benzylphenyl)piperazine; 1-(4-(benzyloxy)phenyl)piperazine;1-(4-phenoxyphenyl)piperazine;1-(4-(4-(methylthio)phenoxy)phenyl)piperazine;4-(4-(piperazin-1-yl)phenoxy)benzoic acid;4-(4-(piperazin-1-yl)phenoxy)benzamide;5-(4-(piperazin-1-yl)phenoxy)picolinamide;1-(4-(3,4-dimethylphenoxy)phenyl)piperazine;4-(4-phenoxyphenyl)piperidine;4-(4-((tetrahydro-2H-pyran-4-yl)oxy)phenyl)piperidine;4-(4-(piperidin-4-yl)phenoxy)piperidine;1-(4-((tetrahydro-2H-pyran-4-yl)oxy)phenyl)piperazine;1-(4-(piperidin-4-yloxy)phenyl)piperazine; and1-(4-phenoxyphenyl)piperazin-2-one; or a pharmaceutically acceptablesalt thereof.
 21. A kit comprising: (i) a compound of formula I:

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein: A is —SO₂— and B is —NH—; or A is —O— or —CH₂— and B is —CH₂—or absent; ring C is a 5 or 6 membered heteroaryl or heterocyclic ring,or a 6 membered aryl ring; E¹, E², and E³ are independently selectedfrom C, CH, and N, wherein one of E¹, E², and E³ may be absent; R¹ andR² are independently selected from the group consisting of H, loweralkyl, hydroxy, amino, aminoalkyl, hydroxyalkyl, haloalkyl, carboxy,—CONH₂, —CON-alkyl, nitrile, —S-alkyl, —O-alkyl, acyl, and oxo; or R¹and R² with the atoms attached thereto form a 5-6 membered fused aryl,heteroaryl, carbocyclic or heterocyclic ring D containing 0-3heteroatoms, where ring D may further be substituted at a position twoatoms away from the juncture with ring C; R³ is independently selectedfrom the group consisting of H, lower alkyl, hydroxy, amino, aminoalkyl,hydroxyalkyl, haloalkyl, carboxy, —CONH₂, —CON-alkyl, nitrile, —S-alkyl,—O-alkyl, acyl, and oxo; R⁴ is selected from the group consisting of H,OH, halogen, and lower alkyl; each R⁵ is hydrogen or taken together areoxo; Y is selected from the group consisting of N and CH; and X¹, X²,X³, and X⁴ are independently selected from the group consisting of CH,C—R⁴, and N; and (ii) instructions for use of said compound forinhibiting PCSK9 in a subject with a metabolic disease.
 22. A method oftreating a patient with diabetes, wherein the method comprisesadministering to the patient a compound of formula I:

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein: A is —SO₂— and B is —NH—; or A is —O— or —CH₂— and B is —CH₂—or absent; Ring C is a 5 or 6 membered heteroaryl or heterocyclic ring,or a 6 membered aryl ring; E¹, E², and E³ are independently selectedfrom C, CH, and N, wherein one of E¹, E², and E³ may be absent; R¹ andR² are independently selected from the group consisting of H, loweralkyl, hydroxy, amino, aminoalkyl, hydroxyalkyl, haloalkyl, carboxy,—CONH₂, —CON-alkyl, nitrile, —S-alkyl, —O-alkyl, acyl, and oxo; or R¹and R² with the atoms attached thereto form a 5-6 membered fused aryl,heteroaryl, carbocyclic or heterocyclic ring D containing 0-3heteroatoms, where ring D may further be substituted at a position twoatoms away from the juncture with ring C; R³ is independently selectedfrom the group consisting of H, lower alkyl, hydroxy, amino, aminoalkyl,hydroxyalkyl, haloalkyl, carboxy, —CONH₂, —CON-alkyl, nitrile, —S-alkyl,—O-alkyl, acyl, and oxo; R⁴ is selected from the group consisting of H,OH, halogen, and lower alkyl; each R⁵ is hydrogen or taken together areoxo; Y is selected from the group consisting of N and CH; and X¹, X²,X³, and X⁴ are independently selected from the group consisting of CH,C—R⁴, and N.
 23. The method of claim 22, wherein PCSK9 in the patient isinhibited.
 24. The method of claim 23, wherein an uptake of LDL byhepatocytes in the patient is modulated.
 25. The method of claim 22,wherein the compound of formula I is formulated as a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier orexcipient.
 26. The method of claim 25, wherein said pharmaceuticalcomposition further comprises a second pharmaceutical drug compound. 27.The method of claim 26, wherein said second pharmaceutical drug compoundis selected from the group consisting of a statin, a cardiovasculardrug, a metabolic drug, and an antihypertensive drug.
 28. The method ofclaim 22, wherein the compound is selected from the group consisting of:N-phenyl-4-(piperazin-1-yl)benzenesulfonamide;N-isopropyl-4-((4-(piperazin-1-yl)phenyl)sulfonamido)benzamide;N-methyl-4-((4-(piperazin-1-yl)phenyl)sulfonamido)benzamide;N-(3,4-dimethylphenyl)-4-(piperazin-1-yl)benzenesulfonamide;N-(3,5-dimethylphenyl)-4-(piperazin-1-yl)benzenesulfonamide;N-(3,5-dichlorophenyl)-4-(piperazin-1-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;N-(naphthalen-2-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(cinnolin-7-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(isoquinolin-7-yl)-4-(piperazin-1-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(quinolin-6-yl)benzenesulfonamide;N-(cinnolin-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(quinoxalin-6-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(pyrido[2,3-b]pyrazin-7-yl)benzenesulfonamide;N-(1H-benzo[d][1,2,3]triazol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(2-methylbenzo[b]thiophen-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(benzo[c][1,2,5]thiadiazol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(1H-indol-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(1H-indol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(benzo[d]isoxazol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(2-aminoimidazo[1,2-a]pyrimidin-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(2,3-dihydrobenzofuran-6-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(2,3-dihydrobenzofuran-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;N-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-4-(piperazin-1-yl)benzenesulfonamide;2-hydroxy-N-phenyl-4-(piperazin-1-yl)benzenesulfonamide;4-(2-oxopiperazin-1-yl)-N-phenylbenzenesulfonamide;2-fluoro-4-(2-oxopiperazin-1-yl)-N-phenylbenzenesulfonamide;N-phenyl-4-(piperazin-1-yl-2,2,6,6-d₄)benzenesulfonamide;N-phenyl-4-(piperidin-4-yl)benzenesulfonamide;4-(piperidin-4-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(pyridin-4-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(pyridin-3-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(pyridin-2-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(thiazol-2-yl)benzenesulfonamide;4-(piperazin-1-yl)-N-(quinolin-7-yl)benzenesulfonamide;N-phenyl-6-(piperazin-1-yl)pyridine-3-sulfonamide;N-phenyl-5-(piperazin-1-yl)pyridine-2-sulfonamide;N-(3,5-difluorophenyl)-4-(piperazin-1-yl)benzenesulfonamide;3-fluoro-4-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;6-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)pyridine-3-sulfonamide;5-(piperazin-1-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)pyridine-2-sulfonamide;N-phenyl-4-(piperidin-4-yl)benzenesulfonamide; and4-(piperidin-4-yl)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide;or a pharmaceutically acceptable salt thereof.
 29. The method of claim22, wherein the compound is selected from the group consisting of:1-(4-benzylphenyl)piperazine; 1-(4-(benzyloxy)phenyl)piperazine;1-(4-phenoxyphenyl)piperazine;1-(4-(4-(methylthio)phenoxy)phenyl)piperazine;4-(4-(piperazin-1-yl)phenoxy)benzoic acid;4-(4-(piperazin-1-yl)phenoxy)benzamide;5-(4-(piperazin-1-yl)phenoxy)picolinamide;1-(4-(3,4-dimethylphenoxy)phenyl)piperazine;4-(4-phenoxyphenyl)piperidine;4-(4-((tetrahydro-2H-pyran-4-yl)oxy)phenyl)piperidine;4-(4-(piperidin-4-yl)phenoxy)piperidine;1-(4-((tetrahydro-2H-pyran-4-yl)oxy)phenyl)piperazine;1-(4-(piperidin-4-yloxy)phenyl)piperazine; and1-(4-phenoxyphenyl)piperazin-2-one; or a pharmaceutically acceptablesalt thereof.